Medicine

Blood clotting

 

VIOLATIONS OF plateled HEMOSTASIS

 

1. Common characteristic of hemostasis system (Hemostasis is very important for our life, because if we are live our hemostatic system is very strong. They are includes in a case of trauma, cutting the vessels etc.)

a) Determine the notion “system of hemostasis” (Hemostasis is the physiologic system, which supports the blood in the fluid condition and prevent bloodless. Hemostasis system vital necessary and functionally connect with the cardiovascular, breathing, endocrine and other systems.)

b) Functional-structure components of hemostasis system (The components of hemostasis are wall of the vessels, blood cells – platelets, erythrocytes, leucocytes, enzymes and nonenzymes components of plasma – clotting and anticlotting substances, fibrinolysis components of hemostasis.)

c) Mechanisms of hemostasis (There are 2 kinds of hemostasis. They are vessel-platelets (primary) and coagulative (secondary) hemostasis. Primary hemostasis activity begin the first after the destroyed of vessels. Secondary hemostasis add after that in case the primary hemostasis do not stopped the bloodless.)

2. Vessel-platelets hemostasis (or primary hemostasis include in clotting first of all after the destroyed the safe of vessel wall.)

 

 

PLATELETS OR THROMBOCYTES

Platelet Function

Platelets are 2 to 4 um in diameter anucleate fragments of megakaryocytes with normal circulating numbers falling between 150,000 and 400,000/uL. Thrombopoietin is the predominant mediator of platelet production, although other inflammatory mediators, such as interleukin (IL)-6 and IL-11, may play a role. Up to 30% of circulating platelets may be sequestered in the spleen and can be released in response to catecholamines. If not consumed in a clotting reaction, platelets are normally removed by the spleen with an average life span of 7 to 10 days. 

In wet preparations of the blood the platelets appear as small (average diameter = 1.5 μm), colorless, moderately refractile bodies that are discoid or elliptical in shape. In stained smears they are round, oval or rod shaped. Platelets do not have nucleus. Their cytoplasm is hyaline, bright blue having azurophilic granules. Young platelets are larger than old ones.

Platelets have small mitochondria, glycogen granules, lipid inclusions and ferritin granules (siderosomes). On the basis of dry weight platelets have 60 % protein, 15 % lipids (phospholipids, arachidonic acid) and 8 % carbohydrate (mainly glycogen, heteropolysaccharides, complexes containing sialic acid). Their major energy source is derived from glucose by glycolysis. Their ATP content is 150 times more than that of RBCs. Their surface has glycoproteins in which there are receptors for thrombin and ADP.

Platelet proteins - About 20 proteins including thrombosthenin, albumin, pre-albumin, IgG, IgM, plasminogen and fibrinogen have been demonstrated in the platelets. Thrombosthenin is identical to actomyosin of muscle; it can be dissociated into two segments, A (actin) and M (myosin). Platelets also have ATP-ase activity including Mg2+-Ca2+ dependent type. The contraction of thrombosthenin underlines the phenomenon of clot retraction and may also be involved in platelet aggregation.

 

Platelet granules - At least 3 types of granules are present in the platelets. Their names along with their contents are given below:

i) Lysosomes; these have endoglycosidase and a heparin-cleaving enzyme.

ii)Dense granules; these have Ca2+; serotonin and ADP.

iii) Alpha granules; these have Von Willebrand factor, fibronectin, fibrospondin and a heparin-neutralizing factor (platelet factor 4).

 

The platelets have been shown to release seven factors that help in blood clotting.

Platelet factor 1 - It has been found to be the same as factor V.

Platelet factor 2 - It is the thromboplastic substance.

Platelet factor 3 - It is a phospholipoprotein, which behaves as thromboplastin.

Platelet factor 4 - It has heparin neutralizing properties.

Platelet factor 5 - It acts as fibrinogen.

Platelet factor 6 -It acts as anti-fibrinolysin.

Platelet factor 7- It is the platelet co-thromboplaslin.

In addition, the platelets also release CPFA and CICA whose roles as activators of factor XII and XI respectively have been mentioned earlier. Platelets also provide surface for the activation of prothrombin to thrombin.

 

STAGES IN PLATELET DEVELOPMENT

 

 

 

1. Megakaryoblast - It is the first cell which can be morphologically characterized and identified to form platelets. It arises, as other blood cells, from the non-specific pluripotent stem cell (CPU). It is 15 to 50 μm in diameter and contains a large oval or kidney-shaped nucleus with several nucleoli. The cytoplasm is scanty and intensely basophilic and has no granules. Mitosis may be seen.

2. Pro-megakaryocyte - It is 20 to 80 μm in diameter. The nucleus is oval or irregular in shape; cytoplasm is more abundant and contains fine bluish granules.

3. Megakaryocyte - This cell is so called because it possesses up to 64 N chromosomes instead of the normal 2 N chromosomes (46) of ordinary somatic cell. This poly-ploidy is brought about by a sequence of events termed as endoreduplication in which nuclear material replicates without cytoplasmic division. It has a diameter of 35 to 160 μm and shows two distinct stages. In the first in which the cell is termed as megakaryocyte without granular platelets, the nucleus is either indented or has multiple lobulations. The cytoplasm is finely and diffusely granular. In the second stage, the cell cytoplasm becomes still more increased in amount and the cell is termed as megakaryocyte with granular platelets or meta-megakaryocyte. The platelets differentiate at the periphery of the cell and when the cell dies, these break off from its cytoplasm to enter the blood stream.

 

In a different nomenclature the megakaryoblast, promegakaryocyte and the mature granular megakaryocyte are called stage I, II and III megakaryocyte respectively.

 

The megakaryocyte occurs in the bone marrow very close to the sinusoidal membrane. It is changed to platelets by two methods: (i) it sends pseudopodia of cytoplasm into the lumen of sinuses through apertures in the sinus membrane. Later these separate from the parent cell and arc swept away by the blood stream as platelets, (ii) The megakaryocyte cytoplasm splits outside the lumen of sinuses, giving rise to 2,000 to 4,000 discrete units, the platelets, which enter the sinuses. The nucleus is left behind and degenerates.

The life span of the platelets is about 10 days in man. The spleen stores them as well as mainly sequestrates the damaged or effete (worn-out by age) platelets. Normally 80 % of the total platelets are in circulation and the remaining 20 % are in the spleen. If the spleen becomes enlarged, then it can store more platelets and this ratio may even be reversed. This may obviously result in a decreased blood platelet count, i.e. thrombocytopenia.

Factors affecting Blood Platelet Count - The average number of platelets in the blood is 250,000 (range being 180,000 to 320,000) per cu mm. Following factors affect the blood platelet count:

1. Age - The count tends to be lower in the newborn especially in prematurely born babies.

2. Menstrual cycle - There is a slight increase on the day of ovulation followed by a progressive fall during the 14 days prior to menstruation. A rapid rise occurs after the start of menses.

3. Pregnancy - There is a slight progressive fall during pregnancy which may fall further during the first stage of labor and on the first and second day after child-birth.

4. Injury - This increases blood platelet count.

5. Adrenaline - It increases platelet count by mobilizing platelets from the spleen, which normally stores about 20 % of the total platelets.

6. Hypoxia - This markedly increases platelet count.

7. Smoking - It tends to shorten platelet survival and produces hyper-aggregability of the platelets.

8. Nutritional deficiencies - Platelet count is low in deficiencies of vitamin B12, folic acid and iron.

9. Thrombopoietin - This substance has been isolated from the blood of a thrombocytopcnic patient. The transfusion of this patient's blood into normal persons resulted in an increase in blood platelet count, i.e. thrombocytosis. It has been shown that if large number of platelets is intravenously administered to a person, then there is a decrease in his own platelet production. On the other hand, removal of platelets from the blood stimulates platelet production. These studies show that some type of regulatory system docs control their production. Erythropoietin, which stimulates erythropoiesis is also believed to produce thrombocytosis.

a) Activation of platelets (To do their function platelets must to activate. In the case of activation the platelets form psevdopodias, change the form. There are 2 groups of activators – the first from platelets and second from another cells, plasma. The outside platelets factors, which are produce in plasma, other cell besides platelets – Villibrandt factor, ADP, epinephrine and norepinephrine. The platelets factors, which are produce by platelets serotonin, ADP, thromboxan A2.)

b) Properties and function of platelets (Quantity of platelets is 180-320 G/L. Diameter of platelets is 1-4 micrometers, thickness – 0,5-0,75 micrometers. They are the little peace of megacariocytes cytoplasm (from one megacariocytes may develop few hundred of platelets). Platelets circulated in blood from 5 to 11 days and than destroyed in liver, lungs, spleen by the cells of macrophagal system. Functions of platelets are: 1. hemostatic function – platelets produce substances, which are secures the hemostasis.

Function of platelets are:

1. hemostatic function – platelets produce substances, which are secure the hemostasis. Its produce 12 platelets factors

1 - proaccelerin,

2- factor, which are increase the speed of development the fibronogen in fibrin,

3 - platelets thromboplastin,

4 - antiheparinic factor,

5 - factor which promote aggregation of platelets,

6 – thrompostenin,

7 – antifibrinolizin,

8 – serotonin,

9 - fibrinstabilising factor,

10 – factor which activate profibrinolisin,

11 – inhibitir of thromboplastin,

12 – antilighting factor.

 

 

Other classiffication of platelets factors. The platelets have been shown to release seven factors that help in blood clotting.

Platelet factor 1 - It has been found to be the same as factor V.

Platelet factor 2 - It is the thromboplastic substance.

Platelet factor 3 - It is a phospholipoprotein, which behaves as thromboplastin.

Platelet factor 4 - It has heparin neutralizing properties.

Platelet factor 5 - It acts as fibrinogen.

Platelet factor 6 -It acts as anti-fibrinolysin.

Platelet factor 7- It is the platelet co-thromboplaslin.

2. Angiotrophic function – provide trophic of endotheliocytes of vessel wall, support structure and functions of microvessels. These function is realize by adgesion of platelets to endotheliocytes and injection the enzymes into the endotheliocytes. For one day near 35 G/L platelets do this function.

3. Transport function – transfer the enzymes, ADP, serotonin and other.

4. Phagocytosis function – the contain of platelets help to kill viruses and antigens bodies.

5. Regeneratory function – platelets have the growth factor, which help to grow the endothelial and muscles cells which are present in the vessel wall.

Its produce 12 platelets factors (1 - proaccelerin, 2- factor, which are increase the speed of development the fibronogen in fibrin, 3 - platelets thromboplastin, 4 - antiheparinic factor, 5 - factor which promote aggregation of platelets, 6 – thrombostenin, 7 – antifibrinolizin, 8 – serotonin, 9 - fibrinstabilising factor, 10 – factor which activate profibrinolisin, 11 – inhibitir of thromboplastin, 12 – antilighting factor).

 

 

Other auther determined such functions of Platelets

1. Role in Hemostasis -The platelets are responsible for the primary hemostasis which is brought about by the formation of the primary hemostatic plug which can effectively stop bleeding from capillaries; small arterioles and venules. Effective primary hemostasis requires three critical events, platelet adhesion, platelet activation and secretion and platelet aggregation.

(A) Platelet adhesion - This means attachment of platelets to non-platelet surfaces, e.g. to collagen and elastic fibers of blood vessels. This process is facilitated by Von-Willebrand factor. This factor becomes attached on one side to the collagen fibrils in the vessel wall, and on the other side to receptors over the platelet surface.

(B) Platelet activation and secretion - This occurs in many steps which are given below:

(a) Binding of platelet agonists, i.e. adrenaline, collagen and thrombin the platelet surface, (b) Activation of phospholipases A2 and C. (c) Released arachidonic acid from the membrane phospholipid. (d) Conversion of arachidonic acid to thromboxane A2, (c) Thromboxane-A2 activates phospholipase-C which liberates still more arachidonic acid from the membrane phospholipid (f) Some inositol triphosphate is also liberated from phospholipids. This stimulate the movement of Ca2+ into the platelet cylosol and the phosphorylalion of myosin light chains. The latter interact with actin to facilitate granule movement and platele shape change, (g) Another product of membran phospholipid is diacylglycerol which brings about secretion of granules. The contents of the granules which are poured into the plasma arc heparinase, Ca2+, adrenaline, kinins, fibrinogcn. factor Va, AMP, thromboxane A2, Von-Willebrand factor, fibronectin, thrombospondin and several other platelet factors including a heparin neutralizing factor-4.

(C) Platelet aggregation or cohesion - The ADP released from the platelets modifies the platelet surface in such a manner that a fibrinogen molecule interacts with specific surface glycoprotein receptors on two adjacent platelets and links the two platelets by a glue-like effect. Aggregation of a large number of platelets results in the formation of small platelet plugs called primary hemostatic plugs or white thrombi; this lakes place within seconds alter injury and the process is called primary hemostasis. It is specially effective in preventing bleeding from small blood vessels such as capillaries, arterioles and venules. It should be noted that in addition to the formation of the primary hemostatic plugs, the platelets also contribute several factors which help blood clotting. However, the platelets required for clotting process are relatively much less and usually mild to moderate thrombocytopenia does not cause blood clotting disorders.

Aspirin and other non-steroid anti-inflammatory drugs inhibit the enzyme cyclo-oxygenase thus inhibiting platelet aggregation. These drugs are being used in the treatment and prevention of thrombolic disorders.

Three more factors have been found to be released during platelet release reaction. These are (i) contact product forming activity (CPFA) which contributes to activation of blood clotting factor XII; (ii) collagen induced coagulant activity (CICA) which helps in the activation of factor XI; (iii) Platelet derived growth factor; it stimulates the migration and growth of fibroblasts and smooth muscle cells within the vessel wall which is an important part of the repair process.

2. Other Functions - (i) Platelets are necessary for the maintenance of the vascular integrity. They seem to donate to the endothelial cells some material essential for their integrity. The platelets may themselves enter the endothelial cells to strengthen them. Platelets also seem to repair small or imperceptible vascular injuries by adhering to the basement membrane. Platelets have been shown to provide glycoprotein which helps in their adhesion to the sub-endothelial collagen.

(ii) Platelets transport all 5-hydroxytryptamine (serotonin) of blood and also carry K+.

(iii) They show slight phagocytic activity to carbon particles, immune complexes and virus particles.

(iv) Contraction of thrombosthenin causes retraction of the clot.

3. Role of Arachidonic Acid Derivatives in Platelet Functions - mammalian tissues the 20-C poly-unsaturated fatty acid, arachidonic acid, converted to cyclic endoperoxide namely PGG2. This reaction is catalyzed t the enzyme cyclo-oxygcnase. PGG2 is converted to PGH2 by the enzyme endoperoxidase. Cyclo-oxygcnase and endoperoxidase are collectively called prostaglandin endoperoxide synthase. The fate of PGH2 is given below.

(i) In the platelets the enzyme thromboxane synlhasc converts PGH2 J thromboxane A2 which is later converted to thromboxane B2; the luuq however, is relatively inert.

(ii) In the arterial wall the enzyme prostacyclin synthase converts PGH2 to PGI2 which is also called prostacyclin.

These two compounds, i.e. thromboxane A2 and prostacyclin possess opposite biological properties. Thromboxane A2 is a powerful vasoconstrictor and promotes aggregation of platelets. As opposed to the actions of thromboxane A2, prostacyclin is a vasodilator and prevents aggregation of platelets. In addition to preventing platelet aggregation, it also has disaggregatory action, i.e. it causes dispersion of any already present platelet aggregates c platelet thrombi. These two substances act through varying the activity of the enzyme adenylate cyclase. For example, prostacyclin activates this enzyme which catalyses the production of 3', 5', cyclic AMP (c-AMP); this in turn activates enzymatic process that leads to the binding of Ca2+ to a Ca-binding protein (calmodulin) in the platelets. This leads to a decreased availability of Ca2+ due to which thrombosthenin can not function properly. This results in a decreased adhesion and aggregation of platelets. On the other hand, thromboxane A2 decreases the activity of the enzyme adenylate cyclase thereby increasing thrombosthenin activity; this leads to more tendency of platelets for undergoing adhesion and aggregation.

4. Role of platelets in atherosclerosis - The essence of atherosclerosis is the formation of atheromalic plaques. Platelets arc believed to contribute to this process. This may be brought about by the release of lysosomal enzymes and other toxic factors from the platelets which injure the vascular endothelium. Platelets also release a growth factor that stimulates proliferation of fibroblasts and migration of monocytes to the injured area. Thromboxane A2 favors while prostacyclin inhibits the development of atherosclerosis. Prostacyclin which can be called a hormone is being used in the treatment of peripheral arteriosclerosis with good results. More recent work has shown that PGI3 and thromboxane A3, which possess one more unsaturated bond than PGI2 and thromboxane A2, are also produced in the body. PCI3 is as potent anti-aggregator of platelets as PGI2 but thromboxane A3 is a weaker pro-aggregator than thromboxane A2. Fish oil is rich in the precursor fatty acid (5, 8, 11, 14, 17-eicosa pentaenoic acid) and its consumption provides both prostacyclin A3 and thromboxane A3. As the latter has weak pro-aggregation effect on platelets while PGI3 has a potent anti-aggregation effect on platelets, the simultaneous presence of both favors anti-aggregation activity of platelets. This has a preventive effect on thrombosis. Eskimos who cat a lot of fish oil have a relatively low incidence of coronary thrombosis.

c) Stages of vessel-platelets hemostasis (1. Shorting spasm of the vessels – vascular spasm duration to 1 minute is caused by catecholamins and other enzymes. Diameter of vessels decrease on ½-⅓. Mechanism of it development determine by secretion of serotonin and thromboxan A2 from platelets and epinephrine from ending of sympathetic nerves. 2. Adgesion of platelets – activation of platelets and stick it to the place of defect in vessel wall. 3. Reverse aggregation of platelets – the thromb which are formed may make way for plasma. 4. Unreverse aggregation of platelets – the thromb which are formed can not may make way for plasma. 5. Retraction of platelets plug – decrease the size of plug, pack down the plug.)

d) Investigation of vessel-platelets hemostasis (1. Calculation of the platelets quantity 180-320 G/L. 2. Determination of duration of capillary bleeding after Duke’s method – to 3 minute in norm. 3. Sample of fragility of capillars – to 10 petechias in norm in a round with diameter 5 centimetres.)

 

 

 

COAGULATION OR CLOTTING OF THE BLOOD

Blood has two remarkable properties; it remains fluid while in blood vessels and clots when it is shed. Both these properties are essential for normal life. The blood contains substances or factors, which favor coagulation (pro-coagulants); it also has substances, which are anti-coagulants. An optimum balance of these two opposing factors is essential for a normal life. The clotting, in essence, is the formation of the insoluble protein fibrin from the soluble plasma protein fibrinogen.

A large number of substances take part in producing fibrin from fibrinogen in the coagulation of blood. The coagulation process actually is the property of plasma though it is commonly termed as clotting of blood. Although a complete understanding of the mode of action of the procoagulants is still not possible, but it can be said that clotting is produced by a complex series of reactions. Once initiated, the whole process proceeds like a chain reaction until clotting is complete. Three methods, which have been much employed for understanding the clotting mechanism are given below.

1. Appropriate techniques by which the clotting process can be stopped at any required stage followed by its re-start.

2. Studies on patients suffering from hemorrhagic diseases.

3. Experimental studies in animals; hemophilia occurs in dogs which have been used for research in this disease.

Blood Clotting Factors - The various factors, which are known to take part in the clotting process in various theories of blood coagulation are given below. These factors have been assigned numbers, which arc written in Roman pattern.

I. Fibrinogen

II. Prothrombin (Thrombin is factor II-a)

III. Thromboplaslin. This is the name given to a substance capable of converting prolhrombin to thrombin. It is present in tissues in an active form, the tissue thromboplastin, which is also called the tissue pro-coagulant material.

IV. Calcium ions.

V. Labile factor, Pro-accelerin, Accelerator or Ac globulin.

VI. It has been found to be the same as factor V; it is now obsolete.

VII. Stable factor, Pro-convertin, Auto-prothrombin-I.

VIII. Anti-hemophilic globulin (AHIG, Platelet cofactor-I. Anti-hemophilic factor A (AHF-A). This is the original compound called factor VIII. However, factor VIII has been found to have three subtypes. The original factor VIII (AHF-A) is now called factor VIII-C, C signifying coagulant action. The other two subtypes are factor VIII V.W. (also called Von-Willebrand protein) and factor VIII R.Ag (protein precipitated by specific rabbit anliserum).

IX. Christmas factor, Plasma thromboplastin component (PTC), Platelet co-factor-II, Auto-prolhrombin-II, Anti-hemophilic factor B.

X. Stuart-Prower factor.

XI. Plasma thromboplastin antecedent (PTA), Anti-hemophilic factor-C, Rosenthal factor.

XII. Hageman's factor, Contact factor, Glass factor.

XIII. Fibrin stabilizing factor, Laki-Lorand factor, Transglutaminase, Pre-fibrinoligase.

 

In addition, the following factors are also associated with blood clotting process.

i) Von-Willebrand factor or the platelet adhesion factor. It is needed for platelet adhesion as well as for activity of factor VIII-C; it is called factor VIII V.W.

ii) Fitzgerald factor; it is the same as high mol. wt. kininogcn.

iii) Fletcher factor; it is pre-kallikrein.

1. Analysis of coagulative hemostasis mechanisms

a) Characteristics of clotting factors (There are 12 clotting factors: I – fibrinogen; II – prothrombine; III – thromboplastin of tissue; IV – ions of calcium; V – proaccelerin; VII – proconvertin; VIII – antihemophylic factor A; IX – Christmas factor or antihemofilic factor B; X – Stuart-Prower factor or prothrombinase; XI – plasma thromboplastin antecedent; XII – Hageman factor; XIII – fibrin stabilizing factor. Some of them are enzymes – II, VII, IX, X, XI, XII,XIII; other  are not – I, III, IV, V, VIII. The vitamin K is necessary for the functional activity of II, VII, IX, X factors.)

b) External mechanism of the first stage (3 factors from the injure tissues go to plasma and interactions with VII factor, the last is activated. VII active factor and IV factors form the complex 1a: III + VII active + IV, which is activated X factor.)

c) Inner mechanism of the first stage (Factor 3 of platelets – platelets thromboplastine – influence on XII factor. Active XII factor + XI is complex 1. Active XI factor activated IX factor. Active IX factor + VIII factor + IV factor is complex 2. Complex 1a and 2 are activate X factor. Factor X active + V + IV formed complex 3 or thrombinasa complex.)

d) Course of the second and third stages (The second stage – formation of thrombin from prothrombin. The third stage is formation of fibrin from fibrinogen. The last stage has 3 period; formation of fibrin-monomers; formation of fibrin S (solubilis); formation of fibrin I (insolubilis). Calcium is necessary for all stages.)

e) Regulation of the clotting mechanisms (Increase of clotting names hypercoagulation, decrease – hypocoagulation. Hypercoagulation may be in a stress cases. It depends on epinephrine, which concentration increased in the cases of stress. Epinephrine increase from the vessels walls factors from which produced prothrombinasa. In cases of big concentration epinephrine should activate XII factor in a bloodstream. It divides fats and fat acids, which have prothrombinase activity. After the hypercoagulation stage may be secondary hypocoagulation.)

 

 

Theories of Blood Coagulation

I. Classical theory of Morowitz (1905-1906) - Blood clotting was considered to take place in two stages.

(i) In the first stage prolhrombin is converted to thrombin by the enzyme prothrombinase, Ca2+ being necessary for this reaction.

(ii) In the second stage the thrombin acts as an enzyme on fibrinogen and converts it to fibrin.

II. Cascade or waterfall theory - For many decades, Morowitz's theory was accepted. But great developments in this field resulted in several new theories, one of which is called cascade or waterfall theory because it involves a cascade of events; it is described below.

There are two systems of clotting, intrinsic and extrinsic, which converge upon what is called the final common pathway.

1. Intrinsic or the blood system - This system is called so, because all factors taking part in the process are derived from the blood itself and it can take place in pure blood (blood not contaminated with tissue juice) kept in a test tube. It is also called contact system because the process starts when blood comes in contact with a foreign surface, e.g. vascular sub-endothelial collagen or even glass. This process takes place in the following six stages. In the first five of these stages limited proteolysis converts an inactive factor to its active form. Each of these steps is regulated by plasma and cellular co-factors and Ca2+. The inactive and active blood clotting factors are distinguished by writing and a respectively after the factor.

Stage No. 1. Three plasma proteins, i.e. Hageman factor (XII), high mol. wt. kininogen and pre-kallikrein form a complex with vascular subendolhelial collagen. Factor XH-i becomes activated to Xll-a, which acceleates the conversion of pre-kallikrein to kallikrein which then accelerates the conversion of still more XII-i to XII-a.

Satge No. 2. Factor XII-a converts factor XI-i to XI-a.

Stage No. 3. Factor XI-a converts factor IX-i to IX-a.

Stage No. 4. Factor IX-a in the presence of factor VIII C, Ca2+ a platelet membrane lipoprotein (platelet factor 3) converts X-i to X-a.

Stage No. 5. Several factors take part in the conversion of prothrombin to thrombin. These include factor X-a, factor V-a, Ca2+ and phospholipids. Although the conversion of prothrombin to thrombin can take place on a phospholipid-rich surface, but it is accelerated several thousand-fold on the surface of activated platelets.

Stage No. 6. Conversion of fibrinogen to fibrin is brought about thrombin by the following mechanism. Fibrinogen is a symmetrical dimer; each half of its molecule has the following structure:

i) Alpha polypeptide joined to a short A-fibrinopeptide.

ii) Beta polypeptide joined to a short B-fibrinopeplide.

iii) Gamma polypeptide.

Fibrinogen can thus be represented by the structure, [Alpha(A), beta(B), gamma]2. Thrombin catalyzes the breakdown of fibrinogen in such a way that a part of the molecule separates leaving behind a fibrin monomer.

[alpha(A), beta(B), gamma]2 → [alpha, beta, gamma]2 (Fibrin monomer) + 2[fibrinopeptide A + B]

However, the removal of fibrinopeptide B is not essential for coagulation. The fibrin monomers undergo polymerization giving rise to fibrin polymers; this process involves formation of hydrogen bonds between fibrin monomers. These fibrin polymers are unstable and the polymerization is readily reversed by inhibitors of H bond formation such as urea. The unstable fibrin polymers are then acted upon by factor XIII, which actually is an enzyme. Factor XIII is initially inactive but is activated by thrombin. It brings about the production of cross linkages between adjacent fibrin polymers. This process involves covalent bond formation between epsilon amino group of lysine and the gamma amide group of glutamine; NH3 is evolved in this reaction. A clot which is much more stable and is insoluble in urea solution is thus produced. Even this fibrin clot is quite soft, but after some time it undergoes retraction during which serum oozes out of it. The platelets are of primary importance in this process of clot retraction. The result is a firm clot that can effectively seal a wounded vessel.

 

2. The extrinsic or the tissue system - This is called so because it needs the presence of tissue juice that contains tissue thromboplastin which is not present in blood. The tissue thromboplastin in the presence of factor VII and Ca2+ activates factor X-i to X-a. Subsequent reactions are the same as described under the intrinsic system and, being common to both the intrinsic and extrinsic systems, are designated as the final common pathway. Because the extrinsic system involves fewer steps than the intrinsic system, therefore it proceeds faster than the latter. For this reason, while the intrinsic system takes 2 to 6 minutes for clotting to take place, the extrinsic system takes as little as 15 seconds to do that.

III. Seeger's hypothesis - This concept basically differs from the cascade theory in that prothrombin and factors VII, IX and X are considered to occur in a single molecular system and not separate from each other. This common molecule is believed to release all these clotting factors during clotting process. A common characteristic of all these clotting factors is that all of them require the presence of vitamin K for their biosynthesis. Factors VII, IX and X are designated by Seeger as autoprothrombin I, II and III respectively. The corresponding active forms of these factors arc called autoprothrombin A, B and C. There are serious objections to this hypothesis as various studies have shown that all these factors arc different and are quite distinct from each other.

Properties of Various Factors Participating in Blood Coagulation

Fibrinogen - It occurs in the plasma in a concentration of 0.35 gram per 100 ml, but is also present in lymph and many tissues. It has a mol. wt. of about 340,000. It is synthesized in the liver. It behaves as a globulin but is more easily precipitated, i.e. by precipitation with 25% ammonium sulfate. The normal plasma has about 15 times more fibrinogen than that required for blood clotting. Its solution is clotted by thrombin; Ca2+ are not needed in this reaction. Afibrinogenemia and dysfibrinogenemia are clinical conditions associated with bleeding; in the former the plasma fibrinogen is absent, while in the latter condition fibrinogen is present but is of abnormal type. Drugs namely Arvin and reptilase are used in therapeutics to control thrombosis; these convert the plasma fibrinogen to fibrin micro-clots that are removed from the circulation by fibrinolysis. In this way, hypofibrinogenemia is produced decreasing the blood clotting tendency. Arvin is obtained from the venom of the Malayan pit viper snake.

Prothrombin - It is the proenzyme, the precursor of thrombin. It contains 2 to 10 % carbohydrate in its molecule and has a mol. wt. of 69,000. Its plasma concentration is 10 to 15 mg per 100 ml.

Thromboplastin - It implies an activity which converts prothrombin to thrombin. All body tissues have this activity and therefore it is termed as tissue or intrinsic thromboplaslin. The brain, lung, placenta and testes are especially rich in it. It is a complex of phospholipids, lipoproteins and cholesterol. Tissue extracts, if injected intravenously, can cause widespread clotting of blood. However, tissue thromboplaslin is not active as such but it needs Ca2+ and factor VII for its activation which normally arc present in blood. Russel viper venom has a strong thromboplaslin activity and is used for slopping bleeding from superficial areas by its local application in diseases like hemophilia.

Calcium - Ca in ionic form, Ca2+, is essential for clotting of blood and it acts at many stages. Ca ions serve to form complexes with lipids, which take part in blood clotting. In health or disease blood has always sufficient Ca2+ for this purpose. In other words, a Ca2+ deficiency is never a cause of a prolonged clotting time in man.

Factor V - It is activated by small amount of thrombin which in turn leads to a greater formation of thrombin. But an excess of thrombin destroys it and causes its disappearance from serum. It is unstable in the citrated plasma. Its congenital deficiency is the cause of parahemophilia, a mild bleeding disorder.

Factor VII - It is stable on storage. It acts as co-thromboplastin in the working of extrinsic system of blood cloning. Its congenital deficiency has been seen very rarely. It has up to 50 % carbohydrate in its molecule.

Factor VIII-C - It is also called platelet cofactor-I and anti-hemophilic globulin. Its deficiency causes the classical hemophilia (now called hemophilia A). Hemophilia is discussed later in detail. This factor is readily inactivated in vitro.

Factor IX - It is also called Christmas factor because its deficiency was first demonstrated in a patient with the surname Christmas whose bleeding disease was named Christmas disease. This disease is also called hemophilia B.

Factor X - It is an alpha globulin present both in scrum and plasma. I deficiency is seen in both sexes equally as a congenital defect.

Factor XI - Its deficiency causes hemophilia C, which is a mild bleeding disease.

Factor XII - It is activated by surface contact and according to the cascade theory, this process initiates the series of reactions leading to blood clotting. Blood deficient in this factor docs not clot in lest tube, i.e. in vitro. If blood taken from a vein (without letting it being mixed with tissue juice) is placed in a lest tube lined with silicone, it does not clot; this is because the silicone layer is smooth and unwettable and does not permit the activation of factor XII for the same reason. Blood also clots much more slowly when placed in polythene tubes as compared to glass tubes. The deficiency of this factor is seen in persons with Hageman's trait, but they do not generally show bleeding tendency. Its additional roles arc the activation of fibrinolytic system and the plasma kinin syslem. It is activated by contact with glass, negatively charged surfaces, collagen fibers, unbroken skin, sebum, long chain fatty acids, uric acid, fibrin, elastin and homocysteine.

Factor XIII - It is the enzyme transglutaminase, whose function has already been discussed. Persons with congenital deficiency of this factor have bleeding tendencies and poor wound healing. Their blood clots all right, but the clot, unlike the normal clot, is unstable and can be solubilized in 5 molar urea or 1 % monochloracetic acid solution.

 

Valuation of clotting

a) Coagulogram (Time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparin time – 50-60 seconds; fibrinolysis – 15-20 %.)

b) Thromboelastography (Thromboelastography is a method of regestration of plugs forming and characteristic of clot by thromboelastograph. The characteristic of clot in thromboelastogramm: a) time of bloods’ beginning clot (from the taking the blood to the first waves of amplitude to 1 mm on thromboelastogramm) – 8-12 minutes; b) time of thromb producing (time of the first waves of amplitude of 1 mm to 20 mm on thromboelastogramm) – 5-8 minutes; c) maximum amplitude (this characteristic of thromb elasticity) – 45-60 mm.)

 

Anticoagulative mechanisms. Fibrinolysis.

1. Common characteristic of physiological anticlotting substances (The tendency of blood to clot is balanced by a number of limiting reactions that tend to prevent clotting inside the blood vessels and to break down any clots that do form.)

a) Properties of antithrombin III (It is the most important anticoagulant in the blood. It inhibits thrombin, factors Xa, IXa, V, XIa, XII. It is basic plasma cofactor of heparin. Very faint inhibitor of plasmin and kallikrein.)

b) Importance of heparin (Blood does not ordinarily clot internally in the body. It is assumed that, unless there is access to injured surfaces, there are not enough thromboplastic substances liberated to convert prothrombin into thrombin and thus start the series of chemical reactions that results in clotting. Even so, additional safeguards are present in antiprothrombic substances such as heparin. This substance was originally found in the liver but is now thought to be produced by large basophilic cells (mast cells) in tissues of various organs. Heparin reduces the ability of the blood to clot by blocking the changeof prothrombin to thrombin. It can also be used to aid in reducing clots in cases in which internal clotting has already occurred. In either case it acts in conjunction with a plasma cofactor. Internal clotting is called thrombosis. The clot, or thrombus, can form in some blood vessel of the arm or leg and do comparatively little harm, but if it should block the blood supply to the brain or to the heart (coronary thrombosis), it can be very serious. Heparin form complex with antithrombin-III and transform it in anticoagulant with the negative action. Activate nonenzyme fibrinolysis.)

c) Role of alpha-2-macroglobulin, alpha-1-antitripsin, protein C (Alpha-2-macroglobulin is very large globulin molecule. It is a similar to antithrombin-heparin cofactor in that it combines with the proteolytic coagulation factors. Its activity is not accelerated by heparin. Its function is mainly to act as a binding agent for the coagulation factors and prevent their proteolytic action until they can be destroyed in various ways. It a faint inhibitor of thrombin, connect with plasmin. Alpha-1-antitripsin inhibits thrombin activity, IXa, XIa, XIIa factors, plasmin and kallilrein. Protein C inhibits VIIIa, Va factors. It activity depend of thrombin and vitamin K concentration.)

d) Functionation of secondary anticlotting substances (Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting. They are antithrombin-I or fibrin and products of fibrinolysis or products of fibrinogen degradation. Fibrin is sorbs and inactivates thrombin and Xa factor. Products of fibrinolysis inactivate ending stage of clotting, IXa factor, platelets' agregation.)

2. Fibrinolytic system (Fibrinolysis is begining with the retraction. The plasma proteins contain a globulin called plasminogen or profibrinolysin, which activated into plasmin or fibrinolysin.)

a) Stages of fibrinolysis

 


active                       prekallikrein

factor XII                activator                          Kinins

 


               Proactivator                 Activator

 

                              Plasminogen                  Plasmin

 


                                 Fibronogen                                 Fibrinogen

         Thrombin                                                           degradation

                                    Fibrin                                       products

 

 

 

b) Mechanisms of fibrinolysis activation (There are 2 mechanisms of fibrinolysis activation: external and internal. Main internal mechanism put in action by XIIa factor. External mechanism stimulated by protein activators of plasminogen, which are produce by vessel wall.)

c) Regulation of fibrinolysis (All processes are direct on increase the clotting mechanism, for example, epinephrine, which are increase in the case of stress. It promote the forming of prothrombinase, activating of XII factor, increasing of fats and fats acids. But after the clotting send up the anticlotting mechanism – hypocoagulation. In norm it has independent regulation. The lysis of blood clots allows slow clearing of extraneous blood in the tissues and sometimes allows reopening of clotted vessels. Reopening of large vessels occurs only rarely. But an important function of the fibrinolysin system is to remove very minute clots from the millions of tiny peripheral vessels that eventually would all become occluded were there no way to cleanse them.)

d) Notion about nonenzymes fibrinolysis (It may be steroid hormons with anabolic function, which are increase producing of fibrinolysis activators by endothelium. Leucocytes ensure function of independent mechanism of fibrinolysis. It limited the size of thromb. Erythrocytes ensure function of independent mechanism of fibrinolysis too.)

3. Functionating of anticlotting mechanisms

a) Role of vessels endothelium in support blood in the fluid condition (1. Smooth surface of vessels endothelium. 2. Negative charge of endotheliocytes and blood cells and that’s why they are push away. 3. Present on the vessels wall thin layer of fibrin which adsorb clotting factors, especially thrombin. 4. Constant presence in blood anticlotting factors in a small doses. 5. Producing by endothelium prostaciclins, which are powerful inhibitors of platelets aggregation. 6. Ability of endothelium to produce and fix antithrombin-III.)

 

THE FIBRINOLYTIC SYSTEM

 

A proteolytic enzyme, fibrinolysin or plasmin, acts on fibrinogen and fibrin causing their breakdown or dissolution by converting them into smaller peptides which are soluble; these peptides are called fibrin/fibrinogen degradative products (FDP) as well as fibrin/fibrinogen split products (PSP). The FDP so produced have several important actions, which oppose hemostasis; in other words they promote bleeding.

 

 

1. They inhibit binding of fibrinogen to platelets and inhibit aggregation of platelets.

2. They inhibit thrombin formation and also destroy any thrombin formed.

3. They prevent fibrin polymerization.

Formation of plasmin - Normally plasmin occurs in blood plasma as its inactive precursor called pro-fibrinolysin or plasminogen. The conversion of plasminogen to plasmin involves cleavage of a single arginine-valinc bond and is catalyzed by an activator which itself occurs in an inactive form namely pro-activator. The pro-activator is changed to activator by enzymes called pre-kallikrein activators, which are derived from breakdown of active factor XII; in other words, the activators of pre-kallikrein are the fragments of active factor XII.

Factors increasing the formation of plasmin - These are discussed below.

1. Fibrinolysokinase - It is present in plasma, many tissues and in many secretions, e.g. saliva, milk and tears.

2. Bacterial enzymes - These include staphylokinase and streptokinase which are produced by staphylococci and streptococci respectively. Streptokinase is used by intravascular infusion for the clearance of thrombo-embolism.

3. Urokinase - It is present in urine and is being used in therapeutics to accelerate fibrinolysis. It is formed in the kidney; its commercial source is the fetal kidney tissue culture. The urinary plasmin produced under its influence is believed to have a role in keeping the renal tubules patent by preventing the deposition of fibrin within them. Fibrin may be derived from the fibrinogen present in trace amount in the renal tubular fluid. In the same way, plasmin activity present in milk, tears and semen serves to keep the corresponding duct systems patent.

4. Cytokinase - It is present in tissue cells, WBCs and platelets.

5. Hormones - Growth hormone and thyroid stimulating hormone (TSH).

6. Miscellaneous - Exercise, adrenaline, hypoxia, histamine, bacterial pyrogens, ischemia, shock, tissue damage and chloroform.

Factors inhibiting plasmin formation - The plasmin activation is inhibited by the following factors.

1. Epsilon aminocaproic acid (Trasylol and tranexamic acid) - It is used in therapeutics to inhibit plasmin system.

2. ACTHl - (Adrcnocorlicotropic hormone).

Control of plasmin activity - A trace of plasmin activity is present even in normal plasma. However, this plasmin activity is kept under check by an opposing factor called anti-plasmin which is 10 times more active than plasmin activity. The anti-plasmin activity is present in alpha-1 and alpha-2 globulins of plasma. When excessive amount of plasmin is formed, it brings about the following two types of effects:

(i) The plasmin gets bound to fibrin clots and breaks them to small fragments which arc cleared by macrophages. This is the physiological action.

(ii) A part of plasmin remains free in the plasma and enters circulation. However, its action is rapidly neutralised by the anti-plasmin. But if plasmin level of the plasma is too high, then it causes digestion of fibrinogen and also of factors V, VIII and XII. This is the pathological action. If this action is very marked, hypofibrinogenemia and deficiencies of factors V, VIII and XII result and lead to hemorrhage.

 

 

 

FACTORS PREVENTING BLOOD CLOTTING WITHIN BLOOD VESSELS

It has already been pointed out that blood must remain fluid within the blood vessels if life has to be normal. The factors, which help in preventing intravascular clotting (thrombosis), are given below.

1. Vascular endothelium - The normal endothelium of blood vessels, has been described to act as a non-wettable surface and docs not allow the activation of factor XII. Similarly the platelets also cannot adhere to normal endothelium. Both these factors prevent the initial steps in clotting. The endothelial cells arc also rich in fibrinolysin activators. In atherosclerosis the normal intima is replaced by a diseased one, and clotting is favored resulting in thrombosis. The endothelium can also be damaged by bacteria, injuries such i as fractures, pressure on veins, etc.

2. Chemical substances in blood - There arc certain substances in the blood, which normally antagonize any tendency towards clotting. These include the following:

(i) Heparin - It is produced by the mast cells of the connective tissue which occur especially in the lung and liver. Heparin is an acidic mucopolysaccharide composed of alternating sulfated D-glucosamine and D-glucuronic acid units. Up to 40 % of its molecular mass is H2SO4. This makes heparin the strongest organic acid in the body. Heparin is formed in small amounts continuously, but its release from the mast cells is greatly increased in conditions like peptone shock and anaphylactic shock. The mechanism of heparin action is manifold. It antagonizes all the stages of blood clotting, e.g. formation of active factor X, thrombin and fibrin. It also inhibits the agglutination of platelets thus decreasing the release of platelet factors. In combination with a protein namely anti-thrombin, it inactivates thrombin. This is probably the most important action of heparin.

Heparin is water soluble. It has mol. wt. of about 17,000. It is much in therapeutics. Excessive doses of heparin lead to bleeding; this cc controlled by protaminc sulfatc intravenously. Heparin is negatively charged molecule and protamine sulfatc being positively charged neutralizes it readily.

(ii) Antithrombins - These arc substances present in plasma which can inactivate large amounts of thrombin very quickly. Thrombin formed during the clotting of only 10 ml of blood can clot the whole body blood. But normally the excess of thrombin is destroyed by antithrombins, and thus the extension of the clotting process to other body regions is prevented; the fibrin clot also adsorb a lot of thrombin and this helps in keeping the clotting process localized. Heparin binds to the lysine residue on the antithrombin molecule, which results in an enhanced activity of the latter. Antithrombins are of great physiological importance. Small amounts of thrombin arc believed to be formed even under normal circumstances, but antilhrombins immediately inactivate it and thus prevent thrombosis. The cervical glands of the medicinal leech (hirudo) contain a substance hirudin which has antithrombin activity. It therefore acts as an anticoagulant and enables the leech to suck blood for a long period from the area where it is applied.

(iii) Anlithromboplastin substances - Blood and body tissues contain substances, which neutralize any thromboplastin released by damage to tissues.

(iv) Protein C-protein-S system - Protein C is a vitamin-K dependent plasma protein. It first occurs in an inactive form. It is activated by thrombin. Its active form acts as a powerful protease; in the presence of a cofactor, thrombomodulin, present on the endothelial surface it digests factors V and VIII and thus clotting process is inhibited. Protein S has the role of accelerating the activation of protein C. These two proteins are believed to have a physiologic role in helping to keep the blood unclotted.

(v) Fibrinolysis - This process has an important role in keeping the blood in a fluid state under normal circumstances. It is believed that micro-thrombi are formed in the blood even normally, but fibrinolysis liquefies these clots. Cellular phagocytosis of small particles of fibrin thrombi is also a protective mechanism.

(vi) Prostacydin - Its role has already been described under platelets.

3. Vigorous circulation of blood - Under normal circumstances this is an important factor in preventing intravascular clotting of blood. This is brought about by not allowing the reactants to accumulate at a certain site, which could initiate clotting. In conditions associated with stasis of blood (bed-ridden patients, congestive heart failure, polycylhemia, hyper-gammaglobulinemia), intravascular clotting is quite common. Use of oral contraceptive pills and pregnancy also produce venous stasis by causing venous dilatation. During vigorous circulation the formed elements of the blood including platelets arc kept away from the vessel wall. Moreover, it facilitates the mixing of the activated clotting factors with their inhibitors. These active clotting factors are also carried to the liver and the reticuloendothelial system, which take them up and destroy them.

 

ROLE OF LIVER AND VITAMIN K IN BLOOD COAGULATION

 

Several factors needed for blood coagulation arc formed in the liver. The formation of four of these factors, i.e. prothrombin and factors VII, IX and X is dependent upon the presence of vitamin K. A deficiency of vitamin K leads to a deficiency of these factors, which may produce a tendency to bleed easily. Substances, which antagonize vitamin K, such as dicumarol, also produce a deficiency of these factors producing hemorrhagic tendency. If the liver is severely diseased, it cannot make these factors at a normal rate and even the administration of vitamin K will fail to exert a beneficial effect on the hemorrhagic tendency. Other factors synthesized in the liver but independent of vitamin K are fibrinogen, factor V and factor XIII. The sites of the formation of factors VIII, XI and XII are .not known, although factor VIII is believed to be produced in the reticuloendothelial cells.

 

 

HEMOSTASIS

Three important mechanisms bring about hemostasis, which means the stoppage of bleeding from an injured area.

1. Local factors - These can be divided into two types.

(a) Vascular spasm - A localized vascular spasm is the first line of defence against bleeding and is brought about by the following changes:

(i) Local spasm of blood vessels which is of myogenic origin. It lasts for upto 20 minutes,

(ii) Vasoconstriction in large vessels which is of reflex (neurogenic) origin. It lasts for only a few minutes,

(iii) Liberation of serotonin, a vasoconstrictor, from the disintegrating platelets.

The early vascular response reduces blood flow and intravascular pressure and therefore facilitates consolidation of the hemostatic plug.

(b) Extra-vascular - These include subcutaneous tissue, muscle and skin, which help in stopping bleeding by covering and thus applying pressure over the wounded area. Wounds of the scalp area bleed more as there is paucity of connective tissue in this area; same is true about Little's area in the nose where bleeding results in epistaxis. Old age, rheumatoid arthritis and excessive use of glucocorticoids also produce atrophy of the subcutaneous tissue with tendency to bleed easily.

2. Role of platelets (platelet plug) - The platelet plug is produced earlier than formation of fibrin and for this reason the platelet plug is said to produce primary hemostasis while fibrin is said to produce secondary hemostasis.

The formation of the platelet plug that stops bleeding from small blood vessels like capillaries is an important defence against bleeding; this process and the additional contribution of platelets to clotting process have already been described. Platelets also release serotonin, a vasoconstrictor.

3. Clotting of blood - This process is very important for maintaining the occlusive plug contributed by the platelets. As soon as blood comes in contact with extravascular tissues, the complex series of reactions leading to blood clotting arc initiated; thrombin formed in this process further activates release of ADP from platelets. The formation of fibrin clot reinforces the platelet plug and the combined fibrin-platelet plug (hemostatic plug) serves to seal the wound more effectively and prevents bleeding after the injured vessels re-open after some time. The clot then undergoes retraction, till after 24 hours its size is only 40% of the original. Platelets and ATP arc needed for the retraction of the clot. The retraction of the hemostatic plug results in a still more effective plugging of the wounded vessels.

 

 

Interaction Between Blood Clotting and Kallikrein Systems - It is quite complex. Both systems affect each other and greatly accelerate blood coagulation.

(i) Active factor XII or its breakdown product acts as an enzyme that increases the reaction, Prekallikrein → Kallikrein.

(ii) Kallikrein in turn greatly increases the activation of factor XII; active factor XII now activates factor XI and thus accelerates clotting process.

(iii) Kallikrein acts on kininogcn to release kinin which by producing vasodilatation brings more blood containing clotting factors to the site of injury thus intensifying the clotting process.

Fate of the blood clot - The initial hemostatic plug contains the platelets and fibrin. But as the platelets undergo autolysis, therefore after 24 to 48 hours the hemostatic plug consists almost entirely of fibrin. In the meantime fibrinolysis starts as the fibrinolytic system is activated and the destroyed leukocytes release proteolytic enzymes.

A small clot may later be completely liquefied by the process of fibrinolysis. In bigger clots a proliferation of blood vessels and connective tissue may take place and after two to three weeks it becomes a fibrous mass; this process is called organization of the clot. The defects in the vessel wall are covered with endothelium. If the defect in vessel wall is small, the endothelium grows from the ends. But when the defect is large, then the endothelial cells are produced by transformation of smooth muscle cells which migrate from the media of the vessel wall.

agents.

Functional element of microcirculation

Microcirculatory part of vascular system performs all blood functions. There are such types of vessels: arterioles, metarterioles, capillaries and venuls. Mean diameter of these vessels is less than 100 mcm. Arterioles, capillary bed venuls and lymphatic capillaries compose functional element of microcirculation. Main processes as blood-tissue exchange or lymph production are performed there. Mean diameter of capillaries is 3-6 mcm. The length of capillary vessel is near 750 mcm. Capillaries perform exchange in surface near 14000 mkm2. Blood flow velocity in capillaries consists near 0.3 mm/s, which permits passing erythrocytes through capillary in 2-3 s.

 

Microcirculatory bed and functional types of capillaries

 

Depending on structure it distinguished three types of capillaries: somatic, visceral and sinusoidal. Capillaries walls are composed from one layer of endothelial cells and basal membrane. Endothelial cells are active elements of capillary bed. Endothelial cell may produce enzymes as antithrombin III endothelial relaxing factor, endothelial contracting factor, which may activate function of hormones and neurotransmitters on vessel's wall or cause some physiological effects by it. It was determined that endotheliocites may contract and become voluminous. Endoteliocytes contain microfibrills, composed from actin, myosin and other contractive elements. Such structures are directed along cell basis and binds to cytoplasm in places of intracellular contacts. When microfibrills contracting two kinds of effects may be produced: both increasing intracellular split after contraction and increasing cell height and its' prominence inside the vessel. Capillary wall has small splits and a lot of pores. In certain organs capillary walls have some specialties. In kidneys glomeruls, intestinal epithelium, capillaries are fenestrated. This specialty permits passing through endothelial cells water, ions and other even rather large molecules as aminoacids or fructose. In red bone marrow, liver and spleen capillaries have interrupted walls, which let passing even blood cells.

 

 

 Common characteristic of erythrocytes resistance

a) Determine the notion “resistance” and “hemolysis” (Resistancy is a property of erythrocytes to be hole in solution with different concentration. Hemolysis is a process of going out of hemoglobin from erythrocytes in plasma. It may be in cases of destroyed the erythrocytes membranes and without destroyed the erythrocytes. Level of osmotic resistance of erythrocytes is the concentration of NaCl in solution in which hemolyse erythrocytes. Minimal resistance of erythrocytes (0,50-0,45 % of NaCl) – in this concentration of NaCl destroyed erythrocytes with the smallest resistance. Maximal resistance of erythrocytes (0,34-0,32 % of NaCl) – in this concentration of NaCl destroyed all erythrocytes. Osmotic resistance of erythrocytes may decrease and hemolysis may be in a higher concentration of NaCl.)

b) Kinds of hemolysis (There are 3 kinds of hemolysis: biological, chemical, physical. Biological causes of hemolysis are poisons of snakes, for example; chemical – strong acids, ether, for example; mechanical – in patient with the pathology of membranes in a case of running, jumping, for example; temperature – higher temperature.)

2. Deformation properties of erythrocytes (Erythrocytes may pass through capillars, which have a smaller diameter than erythrocytes.)

a) Membrane mechanisms (Depends of structure of membrane, it flexible.)

b) Role of erythrocytes content (Depends of structure of hemoglobin.)

3. Physiological analysis of erythrocytes sedimentation speed (ESS) (In a blood which can not clotting erythrocytes sedimentation. In norm erythrocytes sedimentation speed in male is 2-10 mm/hour, in female is 2-15 mm/hour. It necessary for diagnose the inflammatory processes in human organism.)

a) Notion about erythrocytes aggregation (In healthy person erythrocytes aggregation is absent. Erythrocytes aggregation is develop of connection between erythrocytes (small bridges) and forming chains.)

b) Influence of blood components on erythrocytes sedimentation speed (It may be 2 groups of factors – erythrocytes and plasma. Erythrocytes sedimentation speed is increase in a case of decrease the quantity of erythrocytes, decrease pH of blood, quantity of albumins. Erythrocytes sedimentation speed is increase in a case of increase the quantity of hemoglobin, volume of erythrocytes, protein – fibrinogen, cholesterol, gamma-globulins, increase pH of blood.)

c) Influence of physiological condition on erythrocytes sedimentation speed (Erythrocytes sedimentation speed increase in newborn, pregnancy women, in a case of different inflammation processes.)

 

Key words and phrases: platelet plug, agglutinate, clot, retraction, appears, fibrin threads, thromboxan A, growth factor of platelets, fragility, acselerator of thrombin, platelets thromboplastin, antiheparinic, clotting factor, antifibrinolitic factor, antithromboplastic factor, retractosim, platelets cofactor, fibrinstabilising factor, ADP, petechia, capillary bleeding, Duke time, active and inactive clotting factors, coagulogram, thromboelastography, clots creation, thromboplastinal suspension, opalescention, small grains of fibrin, floces of fibrin, threads of fibrin, net, formed by threads of fibrin, uncompact fibrin clot, compact fibrin clot, loose, tight, concentration of fibrinigen, thromboplastin index, recalcification, thromboplastin time, thromboelastogramm, thromb elasticity thromb elasticity, hypercoagulation, hypocoagulation, fibrinolysis, anticlotting factors, hypocoagulation, antithrombin-III, prostaciclins, plasminogen, fibronogen, fibrin, kallikrein, kinins, fibrinogen degradation products, plasminogen, plasmin, thrombin, antithrombin-I, protein C, vitamin K, antithrombin-III

1. How many and what components take place in vessel-platelets hemostasis?

a) 2; vessel’s wall and platelets; b) 3; vessel’s wall, platelets and plasma’s clotting components; c) 3; vessel’s wall, platelets and plasma’s anticlotting components; d) 3; vessel’s wall, platelets and blood clotting and anticlotting components; e) 4; vessel’s wall, erythrocytes, platelets and blood components

2. What is the normal quantity of platelets?

a) (4,0-9,0) x 109/L; b) (16,0-32,0) x 109/L; c) (180,0-320,0) x 109/L; d) (3,7-4,7) x 1012/L; e) (4,0-5,1) x 1012/L

3. Patient S. in Konchalovsky test has 15 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) monocytes; e) platelets

4. Patient P. in Konchalovsky test has 1 petechiars. What cells injures in this patient?

a) erythrocytes; b) leucocytes; c) basophyls; d) platelets; e) nothing

5. In patient K. by Duke method time of bloodless is norm. What is the norm?

a) to 1 minute; b) to 3 minutes; c) to 5 minutes; d) to 10 minutes; e) to 15 minutes

6. In patient D. by Duke method time of bloodless is norm. What is the norm?

a) 0-3 minutes; b) 1-3 minutes; c) 0-5 minutes; d) 1-15 minutes; e) 1-10 minutes

7. What is the time of clotting by Ly-Wait quantity in norm?

a) not more than 3 minutes; b) 1-3 minutes; c) 5-10 minutes; d) to 5 minutes; e) to 10 minutes

8. What is the time of plasma recalcification in norm?

a) 1-3 second; b) to 3 second; c) to 5 second; d) 5-10 second; e) 60-120 second

9. What is the concentration of fibrinogen in norm?

a) not more than 3 g/L; b) 1-3 g/L; c) 2-4 G/L; d) 2-4 g/L; e) to 5 g/L

10. What substances is the secondary anticoagulant?

a) Antithrombin-III; b) Protein C; c) Heparin; d) Antithrombin-I; e) Alpha 2-antitripsin

11. What substances is the primary anticoagulant?

a) Antithrombin-I; b) Antithrombin-III; c) Kinin; d) Kallikrein; e) Globulins

12. From what factors beginning fibrinolysis?

a) III; b) V; c) VII; d) X; f) XII

13. What factor do not take place in fibrinolysis?

a) III; b) V; c) VIII; d) X; e) XII

Real-life situations to be solved:

1. The quantity of platelets in blood is 100,0 x 109/L. Is it norm? What is the clotting mechanism in this patient?

2. The quantity of platelets in blood is 100,0 x 109/L. What is the breach in this patient?

3. The quantity of platelets in blood is 340,0 x 109/L. What is the breach in this patient?

4. Three-years old patient S. has Duke time 2 minutes. Tomorrow will be the operation on his finger. Do this patient need addition medicines, which correct hemostasis?

5. What is you opinion about the hemostasis of patient F. if you know that clotting time is 8 minutes, Dukes time is 2 minutes, quantity of platelets is 190 x 109/L?

6. What are You opinion about the hemostasis of patient T. if You know that in thromboelastogramm time of bloods’ beginning clot – 10 minutes, time of thromb producing – 7 minutes, maximum amplitude – 52 mm?

7. You know that time of clotting by Ly-Wait is 6 minutes; time of plasma recalcification is 65 seconds; thrombotest of V degree; thromboplastin time is 12 seconds; thromboplastin index is 100 %; concentration of fibrinogen is 3,2 g/L; tolerancy of plasma to heparin is 8 minutes; heparin time is 57 seconds; fibrinolysis is 18 %. What is you opinion about the secondary hemostasis of patient L.?

8. You know that time of clotting by Ly-Wait is 3 minutes; time of plasma recalcification is 56 seconds; thrombotest of VII degree; thromboplastin time is 9 seconds; thromboplastin index is 133 %; concentration of fibrinogen 4 g/L; tolerancy of plasma to heparin is 6 minutes; heparin time is 50 seconds. What is You opinion about the secondary hemostasis of patient W.?

9. You know that time of clotting by Ly-Wait is 12 minutes; time of plasma recalcification is 132 seconds; thrombotest of III degree; thromboplastin time is 17 seconds; thromboplastin index is 88 %; concentration of fibrinogen is 2,1 g/L; tolerancy of plasma to heparin is 11 minutes; heparin time is 60 seconds. What is you opinion about the secondary hemostasis of patient J.?

10. What is the difference between primary and secondary anticoagulants?

 Answers for the Self-Control

1. d;  2. c; 3. e; 4. e; 5 b; 6. a;. 7. c;  8. e;  9. d;  10. d;  11. b; 12. e; 13. a

 

1 real-life situation – It isn’t norm. Norm is (180,0-320,0) x 109/L. Clotting of blood is breaches (aggregation of platelets).

2 real-life situation – It is thrombocytopenia – decrease the quantity of platelets.

3 real-life situation – It is thrombocytosis – increase the quantity of platelets.

4 real-life situation – No, they do not, because the Duke’s time is to 3 minutes to all aged groups.

5 real-life situation – The hemostasis system is norm (clotting time is 5-10 minutes; Dukes time is to 3 minutes, quantity of platelets is 180-320 G/L.)

6 real-life situation – It is norm, because in norm time of bloods’ beginning clot is 8-12 minutes; time of thromb producing is 5-8 minutes; maximum amplitude is 45-60 mm.

7 real-life situation – The secondary hemostasis in norm, because in norm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds; fibrinolysis – 15-20 %.

8 real-life situation – The secondary hemostasis in abnorm – it is hypercoagulation, because in norm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

9 real-life situation – The secondary hemostasis in abnorm – it is hypocoagulation, because in norm time of clotting by Ly-Wait – 5-10 minutes; time of plasma recalcification – 60-120 seconds; thrombotest – IV, V, VI degree; thromboplastin time – 12-15 seconds; thromboplastin index – 80-105 %; concentration of fibrinogen – 2-4 g/L; tolerancy of plasma to heparin – 6-11 minutes; heparinic time – 50-60 seconds.

10 real-life situation – Primary anticoagulants are produce and present all time in plasma and secondary anticoagulants form in a case of blood clotting.

Determination of duration of capillary bleeding after Duke’s method

Make a puncture of the skin of the IV finger by sterilized scarification (3-4 mm depth).

In every 30 seconds attach to the place of puncture by filter paper without pressing. Disappearance of red color confirms us about stopping of bleeding.

In conclusion compare your result with physiological norm.

Calculation of platelets

Put a drop of immersion oil on smear of blood. Calculate 1000 of erythrocytes and platelets under 90x objective and 7x-glass of microscope. The contains of platelets in 1 L of blood may be determined by such formula:

X=(a x b) / 1000, where:

X – quantity of platelets in 1 L of blood; a – quantity of platelets during calculation of 1000 erythrocytes; b – quantity of erythrocytes in 1 L of blood.

To valuate the quantity of platelets in blood and draw in pencil the erythrocyte and platelets.

Determination of time of plasma’s recalcification

Put 0,3 ml of calcium solution into the test-tube. Place it into the “water bath” under 37ºC temperature. In 60 seconds add 0,1 ml of plasma and determine the time of clot’s creation.

In conclusion compare your results with physiological normal.

Tromboplastinal test of Quich

Put 0,1 ml of research blood and 0,1 ml of tromboplastinal suspension into the test-tube. Place the mixture into the “water bath” under 37ºC temperature for 60 seconds. Than add 0,1 ml of CaCl2 solution. Determine the time of blood clot’s appearance. Using your result find the tromboplastinal index after this formula:

            tromboplastine time of healthy person

  T1= --------------------------------------------------- 100%

              tromboplastine time of your patient

In conclusion point out what is protrombine time and protrombine index. Compare your results with physiological norm.

Trombotest

Put 5 ml of CaCl2 solution and 0,1 ml of oxalat plasma into the tube. Place into the “water bath” with 37 ºC temperature for 30 minutes.

You can observe 7 levels of coagulation: I – opalescention; II – small grains of fibrin; III – floces of fibrin; IV – threads of fibrin; V – net, formed by threads of fibrin; VI – uncompact fibrin clot; VII – compact fibrin clot.

The first 3 levels can be observed during hypocoagulation; IV, V, VI levels – normal coagulation; VII – hypercoagulation.

In conclusion point out the state of coagulative hemostasis.

Ethanol test

Put on the test-tube 0,1mL of ethanol solution and 0,4 mL of plasma. The content of test-tube mix and stay on the stage at the room temperature during 10 minutes. The gel is forming, when products of fibrinolisis are in the plasma. Test is negative in norm.

Protaminsulfate test

0,1 ml of protaminsulfate solution add to 0,4 ml of plasma. The contains of test-tube mix and stay on the stage. Through 10 minutes we determinate if gel clot is forming. The test is negative in norm.

What is conclusion about results?

Study of fibrinolysis

Water-bath with temperature 38 0C is prepared. The test-tube, which contain 1 ml of blood from experimental animal (rat) stay there. Through every 10 minutes we bent and take the test-tube off water-bath. The criterions of the start of fibrinolisis are decrease of the clot and change the color of serum. It’s necessary to determinate this criterion throught 30 minutes and 1 hour after the start of the study.

To delineated of chenges.

What about do results witness?

 

SOME LABORATORY TESTS DONE FOR INVESTIGATING BLEEDING DISORDERS

 

(1) Clotting time - (Method of Lee and White). Without letting tissue juice to contaminate the blood, venous blood is withdrawn into a clean, dry, all glass syringe with a wide bore needle. A stopping watch is started the moment blood appears in the syringe and blood coagulation time is recorded from the moment. The needle is detached and 1 ml of blood is delivered into each of the four dry, chemically clean glass tubes with a size 10 by 1 cm. These tube should have been previously placed in a container of water (water bath maintained at 37 °C).

After 3 minutes have elapsed, each of these tubes is lifted from the water bath and is tilled very gently. The clotting time is taken when the tube can be inverted without its contents spilling. The clotting time for each tube is recorded separately and the clotting time is reported as the average of the four tubes. The normal clotting time by this method is 4 to 10 minutes. Clotting time is prolonged in hemophilia and other conditions associated with a deficiency of clotting factors. It remains normal in purpura because in this conditions there are no coagulation defects.

(2) Prothrombin time - (Quick's one-stage method). Oxalated plasma of the patient is obtained and to it is added an excess of thromboplastin, which is usually an emulsion of rabbit's brain. Calcium chloride solution is added. The time taken for clotting of the plasma to occur after the addition of calcium chloride is noted; it is termed as the prothrombin time and normally it is 12 to 14 seconds. The prothrombin time is increased in hypoprothrombinemia. However, the test is not specific for prothrombin deficiency and deficiencies of factors V and X and of fibrinogen also increase its duration.

(3) Bleeding time - (Duke's method). When injury to a small vessel produces only a small defect, the platelet plug, i.e. the white thrombus alone can cause hemostasis. This is the basis for the bleeding time that is employed clinically to distinguish hemostatic deficiency caused by abnormalities of blood capillary wall from those caused by coagulation defects. In doing the test a standardized cut is made usually into the skin of the car lobule. The blood, which flows, is absorbed by a filter paper every 15 seconds. When the filter paper remains unstained on touching the wounded area, it shows that bleeding has stopped. The time taken by the bleeding to stop after the cut is made is the bleeding time. This test is simple but crude. In the majority of normal persons, bleeding lime ranges between 1 to 5 minutes. Bleeding time is prolonged in purpuras.

(4) Platelet count - This is normally 180,000 to 320,000 per cu mm of blood. Bleeding usually occurs when the count falls to 50,000 platelets per cu mm or below.

(5) Plasma fibrinogen level - This is found to be low in conditions associated with excessive fibrinolysis. In this condition the presence of fibrinogen degradative products (FDP) in the plasma and urine can be demonstrated.

(6) Assay of clotting factors - Factors V, VIII, IX, etc. can be quantitatively assessed in the blood.

(7) Capillary fragility test - A strong positive pressure (by inflating sphygmomanometercuff) or a negative pressure (by applying suction cups) is applied and the number of minute petechial hemorrhages (petechiac) is counted in the skin; their number is found to be much increased in conditions associated with an increased capillary fragility, e.g. purpuras.

 

METHODS OF KEEPING BLOOD FLUID IN VITRO - In order for the blood to be kept fluid, certain methods are employed.

These include the following:

(i) Addition of heparin. Its mechanism of anticoagulant action has been already described.

(ii) Addition of substances which take up Ca2+, e.g. citrate, oxalate, ethylene-diamine-tetra-acetic acid or EDTA. The last compound is an example of the chelating

 

Alteration of hemostasis system. DIC-syndrome. Hemophilia

The hemostasis violations are classificated acording to: the etiology, the directivity of variations and the mechanism of progressing. The directivity of variations there are hypocogulation and hypercoagulation, acording to the mechanism of violations progressing there are vessel-throbocyte hemostasis disorder and coagulative hemostasis disorder. Hypocogulation is characterized by the reduced of blood capacity to coagulate. All reasons of hypocoagulation are united in four groups: thrombocytopenia, thrombocytopathy, angiopathy, coagulopathy.

 

Hypocoagulation

 

Thrombocytopenia includes a diseases groups, which are characterised by the decrease of thrombocytes  bloodlevel less than 150x109/l. There are the congenital and acquired forms of thrombocytopenias. The congenital thrombocytopenias are mostly followed by the changes of thrombocytes functional properties, that makes it possible to refer these illnesses to thrombocytopathies group.   

The aquired thrombocytopenias are the result of immune and mechanical  damages, the depression  of thrombocytes forming, and increased thrombocytes using.

There are four groups of immune thrombocytopenias:

a) alloimmune- thrombocytes distruction is the result of noncompatibility at one of blood group systems;

b) transimmune- thrombocytes demage can be carried out by mother autoantibodies, who suffers  from autoimmunal  thrombocytopenia, and these immunoglobulins penetrate through placenta and cause the  thrombocytes amount decrease in infant;

c) heteroimmune-thrombocytes damage is the result of their injury by antigenic pattern under the influence of viruses or the appearance of a new antigene or gapten;

d) autoimmune-thrombocytes destruction of is a result  antibodies synthesis against own thrombocytic antigene.   hjm – jkl

Heteroimmune thrombocytopenia are mostly common for children's age, and autoimmune one – for adult. Werlhof's disease (so-called idiopathic purpura) is the one example of the autoimmune thrombocytopenia, the main reason of it is the decrease of immune tolerance to own antigenes. T-suppressors deficit predetermines B-lymphocytes activation and autoimmune process beginning. The reason of suppression function lymphocytes failure at the idiopathic autoimmune thrombocytopenia isn’t know so far, may be this is T-suppressors genetic trouble.

 

 

The possible mechanism provoking autoagression is the alteration of thrombocytes antigene under the influence of drugss, viruses, bacterias. In some cases the bacterial antigenes, probably, have similarity with the thrombocytic antigenes determinants. Macrophages of the spleen plays the main role in pathogenesis of autoimmune thrombocytopenias, they kill of thrombocytes and decrease their number. At heteroimmune thrombocytopenias, the antibodies are synthesized against alien antigene, which are fixed on thrombocytes surface and which have stipulated the alteration of antigenic pattern (this antigene there can be medicines: quinidine, digitoxinum, sulfonilamid drugs, rifampicinum, hypothiazidum, viruses of rubella, chickenpox, influenza and adenoviral infection, vaccine). Thrombocytopenia is dangerous with progressing hematencephalons, gastrointestinal tract bleedings, hematuria

Thrombocytopathies is characterised by hemostasis disorder in the result of platelets dysfunction. The first group is congenital desaggregative thrombocytopathies without any failures of reaction of granules release. This group consist of Glantsman's thrombasthenia (absence of thrombocytes glycoproteins 2а and 3b complex in shells, which are indispensable components  of thrombocytes adhesion stimulators and fibrinogen interaction), essential athrombia, May-Heglin's anomaly, partial desaggregative thrombocytopathies. Signs of this group diseases are the petechias, frequent nose bleedings, menorrhagia, hematencephalon. (film)

The second group is characterized by the failure of granules release, which leads to absence of thrombocytes aggregation during their contact with collagen fibers of basal mambrane and the absence of the second surge aggregation, and as the result thromboxan А2 synthesis, ADP, serotonin, adrenaline, Са2+ disengagement is violated. The key role in the pathogeny of this defect is played by  cyclogenase and thromboxan-synthetase deficit, decrease of membrane phospholypase activity. Clinical signs are petechias, mild appearance of ecchymoses, nose and gem bleedings are possible.

The third group are the result of thrombocytes disability to store and to select granules content (ADP, serotonin, adrenaline, factor IV) at hemostasis. One of examples is the Herdimansky's-Pudlac's disease, which is characterized by not dangerous bleeding. Another example is TAR-syndrome (pathology of megacaryocytic-thrombocytes with bones anomalies).

The fourth group of  thrombocytopathies are caused by failures of adhesive and agregational thrombocytes (different variants of Willebrand's and Willebrand's-Urgens' diseases - thrombocytes disfunction is the result of Willebrand's factor deficit;  Bernar's-Syle's syndrome, which is the result of megacaryocytes and thrombocytes anomaly, suhc as the increase blood platelets sizes, the absence of glycoprotein 1 in megacaryocytes and thrombocytes cytoplasmic membrane, that being indispensable for the interaction with Willebrand's, V and XI factors. Severe danger even of mild of this disease arises during sexual maturation of the girl (juvenile menorrhagias arise) and at labours.

The fifth group are caused by the deficit and the decrease accessibility of factor III (Boye’s and Oven’s thrombocytopathies). The thrombocytes pathology is characterized by the deficit of membrane phospholipids, that activate of factor III, so normal structural modification of membranes doesn’t take place during adhesion andaggregation  of thrombocytes.

Wiscott-Oldridge's syndrome is the example of the sixth group thrombocytopathies. A reason of thrombocytes pathology is the low content of dense and alpha granules, the small conservation of ATP, ADP, serotonin and reductants of alpha granules, decrease of thrombocytes adhesive properties.

The prognosis for life thus is unfavorable. The majority of the aquired thrombocytopathies are characterized by the complication of pathogeny. Only some medicines and toxins  (conditioned by aspirin) have legible and stable functional marker. Aquired thrombocytopathies arise at acute leukoses (blastal surrounding provokes disorder of thrombocytes maturation). Thrombocytes dysfunction and hemorrhagic syndrome can be immune inhibition. Immunethrombocytopathies  are coused by the capacity of antibodies to damage cytoplasmic membrane and to lock up the receptors. The В12-deficient anemia is also called thrombocytopathy, which is characterized by the disorders of granules release reaction. Thrombocytopathies can arise at uremia, diseases of the liver. Very often the applying of medical drugs can be the reason of the thrombocytes functions disorder. However the mechanism of many medicines action is well studied and this enables to distinguish the following links of pathogeny of thrombocytes activity disorders: 1) suppression of thromboxan А2 synthesis, such operating mechanism is characterized for phospholypase inhibitors, which violate the arachidonic acid synthesis (сhinacrinum, glucocorticoids), for cyclogenase inhibitors (acetylsalycilic acid, indomethacinum, butadionum, ibuprofenum, naproxenum), for thromboxan-synthetase inhibitors (prostacyclinum, imidazole); 2) the decrease of thrombocytes  cAMP level, such mechanism of action is characterized for stimulators adenilatcyclase (prostacyclinum, prostaglandinum Е), for inhibitors of phosphodieterase, which conduce the cAMP degradation (dipiridamolum, papaverine, euphilinum, flavonoids), for stimulators of prostocycline synthesis, (anabolic steroids, niacin); 3) infringement of Ca2+ transportation (verapamilum, corinfarum).

Vassopathies (angiopathies) are the diseases, which are characterized by the bleeding in the result of vascular wall pathology. All congenital vassopathies, despite their variety, are united by the same pathology - congenitalinferiority and improper development of the connective tissue, including vessels subendothelium. Congenital angiopathies are presented by heritable hemorrhagical teleangioectazias, diffusal trunk angiokeratoma, heritable thrombocytopenic microangiomatosis etc.

Bleeding is the basic performance of this disease and is conditioned by the low resistance and easy vunurability of a vascular wall, by very gentle stimulation in these adhesion sections by both the thrombocytes aggregation and blood coagulation. Most often bleedings nose start, however possible are profuse and sometimes fatal bleedings from teleangioectazias of bronchi and gastrointestinal truck, sometimes brain and internal organs hemorrhages are registered.

The group of acquired (secondary) angiopathies (vascular purpuras) includes mostly dermal bleedings, which arise in the result of exogenic or endogenic vessels injuries. There are idiopathic, stagnant (orthostatic), atrophic (dystrophic), neurogenic, mechanical and other acquired vassopathies. The etiology of idiopathic angiopathies is not known (example is the idiopathic hemorrhagic Caposhi’s sarcoma - reticulihystiocytic system malignant tumor with skin bleeding in the result of vessels hemostasis failure). The stagnant angiopathies can be caused by chronic heart failure, local venous insufficiency (Klots' haemostatic dermatitis, Favr-Racusho's dermatitis) and are the result of long-time tissues hypoxia and vassal dystrophy.

 

 

Steroid purpura is the example of atrophic-dystrophic angiopathies, it arises after the durable treatment by glucocorticoids, which suppress the fibroblasts proliferation, decrease the collagen and mucopolysaccharides synthesis that predetermines the dot hemorrhages.

Vessels dystrophy and the following bleeding can be the result of vascular walls damage by immune complexes (Shanline-Genokh’s hemorrhagical vasculitis). The vit. C deficit (scorbutus) also promotes the infringement of collagen fibres synthesis (they ensure the continuity of capillary endothelial covering) and is the reason of bleeding in pericappilar spaces (in fascia, aponeurosis, fatty tissue, muscles, joint cavities, in very severe cases the hemothorax and hemopericardium can develops.

 

Coagulopathy is the example of hypocoagulation in the result of coagulative blood system pathology development. There are primary (hereditary) and secondary (aquired).

The primary coagulopathies are divided on such group as:

1) failure of internal mechanisms prothrobinase activity forming (hemophilia A – procoagulant unit of factor-VIII deficit, hemophilia B – factor-IX deficit and hemophilia C –  factor-XI deficit, Willebradn’s disease, Hageman’s defect –  factor-XII deficit),

2) failure of external mechanisms prothrombinase activity forming (hypoproconvertinaemia – factor - VII deficit),

3) the combined failure of external and internal mechanisms prothrombinase  activity forming (parahemophilia –factor-V deficit),

 4) failure of final stage blood coagulation (hypofibrinogenemia, dysfibrinogenemia).

The patients’ number, which suffer hemophilia A, B, C and Willebrand’s disease occur more often in clinical practice. The hemophilia A is the result of heritable deficit or molecular anomaly of VIII-factors’ procoagulative part, and is characterized by the coagulative hemostasis failure. Normally factor-VIII circulates in blood in the large molecular protein form and consists of a subunits series: glucoprotein having procoagulative property (VШ); glucoprotein with property to cause of thrombocytes adhesion (Willebrand's  factor - VШ:W), ristomicine-cofactor (VIII:R-cоf.), and also antigenic markers of VIII:K and VIII:R-cof.. Activity of both factor VIII:K and VШ:W is being decreased at the decrease of complex structure mass. Synthesis of  factor-VIII   all components is controlled by X-chromosome. The gene of hemophilia is recessive, thus men are suffered mostly (woman, having the second normal X-chromosome, as a rule, don’t suffer from bleeding, but the VIII-factor activity is reduced in twice and this fact should be taken into consideration surgical operations referring to mothers, sisters and especially daughters the person,  which is ill with hemophilia. The hemophilia A сan be caused by poor synthesis of VIII:K factor, thus it’s antigen isn’t discovered in patients plasma (so-called antigen negative hemophilia or hemophilia A-). In other cases antigen activity of VIII:K factor prevails the activity of this component, as the result of   abnormal VIII:K factor synthesis (so-called antigen positive hemophilia A or hemophilia A).

Clinically the hemophilia A is manifested by hemorrhages in major joints of finitenesses, deep hypodermic, intermuscular and intramuscular hematomas, massive and prolonged posttraumatic bleedings. Man, who have cariotype 46,ХhУ and women, who have cariotype 46,ХhХh or 45,ХhО suffer with hemophilia A, but woman, who have cariotype 46,ХХh are the carriers only. 

The Willebrand's disease is the example of autosomal heritable coagulopathy. This is not single disease, but a group of related of hemorrhagic diathesises, which are caused by the infringement of synthesis or the quality anomalies of VIII:W factor. The great number of Willebrand's disease variations is the proof of factor-VIII structure complication. The patients frequently have hypodermic hematomas, parent bleedings with women can be last 15-25 days, are hardly treated, the hemorrhages in large joints are possible. The heritable factor-IX  deficit is called hemophilia B or Cristmas’ disease; it is inherited by the recessive type and is joined with X-chromosome, however the structural gene of the factor-IX  is localized in the other  end of this chromosome and isn’t connected with the gene of VIII: K factor.

Hemophilia B is antigene positive (hemophilia B+) and antigene negative one (hemophilia B-) Such patients have caryotypes 46,XhY, 46,ХhХh, 45,ХhО or 46,XX/45,XhO). Hemophilia B is identical to hemophilia A by clinical developments, by gravity and complications. These hemophilias are distinguished only by the results of laboratory studies. The hemophilia C is the example of autosomal heritable coagulopathies, which caused by the factor-XI deficit.

 

The acquired coagulopathies, as a rule, are caused by the complex infringement in the coagulative system and have much more complicated pathogeny, than the heritable ones. The insulated deficit of the some coagulation factors occurs not often (amiloidosis provokes factor-X deficit, factor-VIII deficit may be result of its immune inhibition at the antigenic noncompatibility of the mother and the fetus. The simultaneous deficit of several factors is frequent in clinical practice. Hemorrhagic syndrome, as a result of   of K-vitamin dependent coagulation factors deficit, is characterized by the disorder of synthesis in hepatocytes and the decrease of factors VII, X, IX and II blood concentrations. Reasons of hemostasis violations and poor vitamin K synthesis can be dysbacteriosis, profusal diarrhea, enteropathies, failures of this liposoluble vitamin suction at bile deficit (mechanical icterus), failure of the final stage vitamin-K dependent coagulation factors synthesis (process of their decarboxilation) due to displacement of vitamin K from the metabolism by competitive antagonists anticoagulants of the indirect action, dangerous liver destructions. The depression of the VII, X, IX, XII factors activity arises thus in succession, this depends on the different lifetime of these factors in blood. the medical drugs using can be complicated by coagulopathies. This happens if the drugs have the anthithrombical activity and are overdosed (heparin especially).

Hypercoagulation.  DIC-syndrome

Hypercoagulation is the organism state characterized by excessive activation of coagulative blood systems. The examples are thrombosis and dessiminative intravascular blood coagulation syndrome (DIC-syndrome). Most often DIC-syndrome arises at the development of infections (especially generalized one); at  septic states coused by bacteriemia or virusemia, including at abortions, after labors, at a long-term vessels catheterization; at shock (traumatic, hemorrhagical, anaphylactic, cardiogenic, septic, in conditions of septic shock the acute DIC-sindrome is registered in 100 % of cases); at traumatic surgical operations; at long-term usage of an artificial blood circulation apparatus; at all terminal states the DIC-syndrome (in 100 % of cases); at acute hemolysis; at anticipatory flaking-off of placenta, at thermal and chemical burns, at immune diseases, at allergic reactions.

The main DIC-syndrome mechanisms are the following:

1) the activation of thrombocytic and coagulative hemostasis components by the endogenenic factors - tissues thromboplastin, factors of tissues and blood cells disintegration, leukocytic proteases, factors of endothelium injured;

 

2) the activation of hemostasis system by such exogenic factors as bacterias, viruses, medicines, snake poisons;

3) the injure of vascular endothelium, followed by the decrease its antithrombical potential;

4) the dissipated intravascular blood coagulation, thrombocytes and erythrocytes aggregation with the formation of many microclots and the block of microcirculation;

5) deep dystrophic failures in organs-targets, the failures of their function;

6) circulatory disturbances which predetermine tissues hypoxia, hemocoagulational  shock, acidosis, microcirculation failure caused by the disability of the organism to promote the capillary hemodilution process, and stop blood flow;

7) the development of the consumption coagulopathy followed by complete disability of blood to coagulate, the exhaustion of anticoagulative  mechanisms (deficit of antithrombin III and protein С), component of fibrinolytic and calicrein-kinin systems;

8) the secondary high-gravity endogene intoxication by toxic substances of proteolysis and tissues destruction.

The key role in a pathogeny of a DIC-syndrome is given to the increase of thrombinum concentrationin (hypethrombinemia), to the exhaustion of hemocoagulational potential.

The main initiator of the coagulation process is the tissues thromboplastin, that comes into the blood from the injured tissues and endothelium. Activated monocytes provoke to produce tissues thromboplastin and this mechanism plays an important role in DIC-syndrome pathogeny at virusemia, at endotoxemia, at immune diseases (activated monocytes start to produce partially activated such factors as VII, X, IX, II. The thrombocytes aggregation and their using for the thrombforming is the obligatory component of DIC-syndrome pathogeny. The erythrocytes at DIC-syndrome are injured, their lifetime in blood is shortened and the intravascular hemolysis appears. This process activates the blood coagulation because much ADP and other agents from injured erythrocytes came into the blood, promotes thrombocytes aggregation, conduces the DIC-syndrome progressing, and microcirculation disorder in tissues. The very important pathogenetic link of this pathology is the activation not only the system of blood coagulation, but also of such plasma proteolytic systems, as fibrinolytic, calicrein-kinin and complement ones. There of the imagination about the acute DIC-syndrome as about the "humoral protease detonating" was developed in the result of which the patients’ blood is filled up with the great amount of proteins disintegration metabolites. These substances can damage a vascular wall; promote bleeding and secondarilly strendhen blood coagulation. DIC-syndrome progressing provokes the decrease of anticoagulants concentration, especially antithrombin III, which is the coagulation enzyme factors inhibitor and protein C, which is the not enzyme factors (f.-VIII and f.-V) inhibitor. Similarly the fibrinolytic components (precallicrein, kininogene) are utilized.

The hemorrhagic syndrome at these conditions is the result of coagulative blood properties failure, namely:

1) the anticoagulative action of toxic substances,

2) usage of the factors VIII and V;

3) failure of thrombocytic hemostasis in the result of hypoxia;

4) toxic influence on a vascular wall of proteolysis products;

5) the decrease in blood of the most active thrombocytes and their block by the fibrin dissociation products.

Thrombocytopenia and thrombocytopathy, which arise at this condition, is the important bleeding factor.

Gravity of DIC-syndrome depends on infringement of microcirculation in organs. Constant satellites of DIC-syndrome are shock lung, acute renal insufficiency and other organ failures. Their development is the result of massive capillary block by fibrin clots and blood cells aggregates. Hematocrite capillary blood index increases until 0,45 - 0,55 l/l at DIC-syndrome. The indicated failure and microvessels thrombosis plays the key role in the development of stasis, hypoxia and organs dystrophy. The current of DIC-syndrome can be acute, lingering, the relapsing, chronic, and latent. Stages of DIC-syndrome are following: the first stage - hypercoagulation and thrombocytes aggregation; the second stage - consumption coagulopathy and thrombocytopathia; the third stage - hypocoagulation; the fourth stage - recovery or consequences and complications (at the unfavorable current the death of the patient is possible). By the gravity of the current DIC-syndrome  may be acute, subacute and chronic.

The main signs of DIC-syndrome are hemocoagulative or mixed shock (at acute form), failure of hemostasis (thrombosis and hemorrhagia), hypovolemia, anemia, disfunction and dystrophy of organs, metabolic disorders. Hemocoagulative shock is the result of microcirculation violations in organs and tissuel hypoxia, the formation of proteolysis toxic substances. This is characterised by the decrease of arterial and central venous pressure, by organs microcirculation violations and acute organs functional insufficiency (acute renal or hepatorenal insufficiency, shock lung). The development of profusal bleeding promotes the transformation of hemocoagulative  shock into the hemorrhagic one.

There are different phases at hemostasis failure - from hypercoagulation up to hypocoagulation (at first we can observe massive thrombogenesis, but then thrombogenesis depression and bleeding). The thrombocytopenia and thrombocytopathy in this condition is the result of great number microthrombuses forming, of the thrombocytes injury and returning to circulation of degranulated thrombocytes. Hemorrhagic syndrome (bleeding) mostly arises at acute DIC-syndrome in hypocoagulation stage. The main pathogenetic mechanisms of bleeding are pathological influence on vessels of toxic proteolysis substances, the failure of thrombocytes angiothrophical function, thrombocytopenia, thrombocytopathy of consumption, fibrinolytic system activation.

The microcirculation block predetermines the disfunction and dystrophy of organs. The organs-targets (lung, kidneys, intestine) suffer mostly. The combined forms of organ violations are more difficult, for example, the combination of pulmonary and renal insufficiencies. The stomach and intestine also belong to the group of organs, which are frequently damaged at a DIC-syndrome. Microcirculation violations provoke mucous membrane dystrophy and profusal bleedings. In complicated cases the failures of cerebral circulation, of paranephroses, of pituitary body, and liver are possible.

 

 

Hemostasis

      Hemostasis - stoppage of bleeding in a quick & localized fashion when blood vessels are damaged

    Prevents hemorrhage (loss of a large amount of blood)

      Three major steps of Hemostasis:

1.     Vascular spasm

2.     Platelet Plug Formation

    Aggregation and adhesion of platelets

3.     Blood Clotting

    Fibrin threads entangle platelets and RBCs to form blood clot

 

Vascular Spasm

      Damage to blood vessel stimulates pain receptors

      Reflex contraction of smooth muscle of small blood vessels

      Can reduce blood loss for several hours, allowing other mechanisms to take over

      Effective only for small blood vessels or arterioles, not major arteries

 

Platelet plug formation

      Platelet Plug Formation steps:

1.     Platelet Adhesion

    Platelets stick to exposed collagen of vessel

2.     Platelet Release Reaction

    Platelets extend projections

    Platelets release Thromboxane A2 Serotonin & ADP activating other platelets

3.     Platelet Aggregation

    Platelets stick together forming a platelet plug

1) Platelet Adhesion

      Platelets stick to exposed collagen underlying damaged endothelial cells in vessel wall

 

2) Platelet Release Reaction

      Platelets activated by adhesion

      Extend projections to make contact with each other

      Release Thromboxane A2 Serotonin  & ADP activating other platelets

    Serotonin & Thromboxane A2 are vasoconstrictors decreasing blood flow through the injured vessel.

    ADP causes stickiness

 

3) Platelet Aggregation

      Activated platelets stick together and activate new platelets to form a mass called a platelet plug

      Plug reinforced by fibrin threads formed during clotting process

Coagulation

 

      A set of reactions in which blood is transformed from a liquid to a gel

      Coagulation follows intrinsic and extrinsic pathways

      Common Pathway:   The final three steps

    Prothrombinase (Prothrombin activator) is formed from Factor X

    Prothrombinase converts Prothrombin into Thrombin

    Thrombin catalyzes polymerization of Fibrinogen into a Fibrin mesh

 

Two Pathways to Prothrombin Activator

      May be initiated by either the intrinsic or extrinsic pathway

    Triggered by tissue-damaging events

    Involves a series of procoagulants

    Each pathway cascades toward Factor X

      Once Factor X has been activated, it complexes with Ca+2 ions, PF3, and Factor V to formProthrombin activator (Prothrominase)

 

Coagulation Pathway

      Prothrombinase & Ca+2 catalyze the conversion of Prothrombin to Thrombin

      Thrombin & Ca+2 catalyze the polymerization of Fibrinogen into Fibrin

    Insoluble fibrin strands form the structural basis of a clot

    Fibrin causes RBCs and Platelets to become a gel-like plug

      Thrombin & Ca+2 also activate Factor XIII (F13) that:

    Cross-links fibrin mesh

    Strengthens and stabilizes the clot

 

Clot Dissolution

      Inactive plasminogen becomes plasmin, a fibrinolytic enzyme

      Plasmin dissolves small clots at site of a completed repair

      Clot formation remains localized

    blood flow disperses clotting factors

      Basophils release heparin (anticoagulant), preventing inappropriate clots

 

Intravascular Clotting

      Thrombosis

 

Clot (thrombus) formed in an unbroken blood vessel

    Attached to rough inner lining of blood vessel

    Blood flows too slowly (stasis) allowing clotting factors to build up locally & cause coagulation

    May dissolve spontaneously or dislodge & travel

      Embolus – free floating clot in the blood

      Low dose aspirin blocks synthesis of thromboxane A2 & reduces inappropriate clot formation,

    Helps to prevent strokes, myocardial infarctions

 

Platelet Disorders

 

Bleeding time is generally very prolonged in patients with uremia, signifying a major defect in platelet function, which improves after dialysis. A number of dialyzable platelet-inhibitory factors have been shown to inhibit platelet function. Furthermore, uremic platelets synthesize less thromboxane A2, and the blood vessels taken from patients with uremia produce greater quantities of platelet-inhibitory prostaglandin. Nitric oxide produced by the endothelial cells inhibits platelet function. Because the prolonged bleeding time and the hemostatic abnormalities are partly corrected by red blood cell transfusion or erythropoietin therapy, the failure of hemoglobin to quench excess nitric oxide synthesis has been suggested as partly responsible for the platelet dysfunction.

Frequency:

In the US: Inherited hemostatic disorders are relatively rare. The prevalence of von Willebrand disease has been estimated at 1 case per 1000-5000 individuals. Acquired hemostatic disorders are common, and ITP is one of the most common autoimmune disorders. It occurs in 2 distinct clinical types, an acute self-limiting form observed almost exclusively in children (5 cases per 100,000 individuals), and a chronic form, observed mostly in adults (3-5 cases per 100,000 individuals) and rarely in children.

Sex:

Unlike hemophilia, most inherited disorders of platelets are not X-linked and are equally distributed in both sexes.

Chronic autoimmune thrombocytopenia is more common in females than in males.

Acute ITP is observed equally in both sexes.

History:

History and physical examination findings help distinguish between primary and secondary hemostatic disorders and whether the disorder is inherited or acquired.

Epistaxis is common in individuals with primary hemostatic disorders, but it is also common in healthy individuals. Details about the frequency, duration, packing requirement, and prior treatment (cautery or transfusion) are helpful for assessing the severity of bleeding.

Bleeding gums is a common symptom in persons with primary disorders of hemostasis. Bleeding could be spontaneous or could be associated with brushing or flossing.

Bleeding from tooth extractions is possible. A molar tooth extraction is a traumatic procedure. Uneventful extraction of a molar is unlikely in a patient with a severe bleeding disorder.

Hemoptysis, hematemesis, hematuria, hematochezia, and melena are rarely the initial symptoms of a bleeding disorder. However, these may be exacerbated by an underlying bleeding disorder.

Menstrual history is important. Metromenorrhagia is often observed in individuals with primary hemostatic disorders. This is especially common in patients with von Willebrand disease and is often exacerbated by the NSAIDs used to treat dysmenorrhea.

Bleeding after childbirth may be the first manifestation of a mild bleeding disorder.

Bleeding in the joints is the hallmark of hemophilia and other secondary hemostatic disorders.

Details of previous surgery, including the amount of blood transfused, if any, are helpful.

In males, excessive bleeding following circumcision is often the initial manifestation of a congenital bleeding disorder.

Delayed bleeding from the umbilical stump is characteristic of a factor XIII deficiency.

Defective wound healing is observed in individuals with a factor XIII deficiency and abnormal fibrinogens.

Medication history findings may be helpful because aspirin often accentuates a preexisting bleeding disorder. A history of previous iron therapy for anemia may be useful.

Bruising is common in individuals with a platelet disorder.

·                     Petechiae are pinpoint hemorrhages (<2 mm) in the skin, and purpura (0.2-1 cm) and ecchymoses are larger hemorrhages. The purpura is not palpable, in contrast to the palpable and sometimes tender purpura observed in patients with vasculitis. Initially, purpura tends to form in the areas of increased venous pressure, such as the legs. Petechiae and purpura may develop following the application of a sphygmomanometer.

·                     Splenomegaly is not observed in the typical patient with ITP. The spleen can engulf platelets and be several times normal size without becoming palpably enlarged.

·                     Hemarthrosis and deep muscle hematomas are unusual in patients with primary hemostatic disorders.

Causes:

·                     Platelet defects can be considered either as a decreased number of platelets (thrombocytopenia) or as defective platelets. Platelet aggregation tests are useful in differentiating various disorders of platelet function. Spurious thrombocytopenia can occur due to aggregates forming in the specimen. Also, dilutional thrombocytopenia may occur in situations of fluid replacement or blood component replacement without platelet support. In all cases of thrombocytopenia, the peripheral blood smear must be reviewed to confirm the thrombocytopenia. This review is crucial.

·                     Thrombocytopenia can be further divided into increased destruction or decreased production. Thrombocytopenia resulting from increased destruction occurs either by an immune mechanism or increased consumption. Platelets are consumed intravascularly by the activation of the coagulation process (diffuse intravascular coagulation [DIC]) or by deposition on damaged endothelial cells (microangiopathy). Production defects result from those diseases that cause bone marrow failure, such as aplastic anemia, infiltration by leukemia or another malignancy, fibrosis or granulomatous disorders, or tuberculosis.

·                     Functional disorders of platelets can be inherited (rare) or acquired (common).

·                     Causes of thrombocytopenia related to increased destruction include (1) immune thrombocytopenias (eg, autoimmune, alloimmune, drug-induced) and (2) increased consumption (eg, DIC, TTP).

·                     Causes of thrombocytopenia related to decreased production include bone marrow depression.

·                     Disorders of platelet function are as follows:

o                                            Disorders of platelet adhesion (von Willebrand disease, Bernard-Soulier syndrome)

o                                            Disorders of aggregation (Glanzmann thrombasthenia)

o                                            Disorders of secretion

o                                            Disorders of thromboxane synthesis

o                                            Acquired disorders of platelet function (drugs, eg, aspirin, NSAIDs, alcohol)

o                                            Uremia

o                                            Paraproteins

o                                            Fibrin degradation products

o                                            Myelodysplasia or a myeloproliferative syndrome

Lab Studies:

·                     Peripheral smear

o                                            Careful examination of the peripheral smear is essential in a patient with thrombocytopenia.

o                                            Spurious thrombocytopenia due to platelet clumping or platelets adhering to neutrophils (platelet satellitism) can be seen in the smear 

Platelet-associated immunoglobulin G

o                                            The autoantibodies responsible for autoimmune thrombocytopenia do not induce complement-mediated lysis. Furthermore, when platelets are destroyed in the circulation, they internalize plasma proteins, including immunoglobulin. Platelets also have low affinity to the crystallizable fragment (Fc) receptor, FcgRIIa, that binds immunoglobulin.

o                                            In patients with autoimmune thrombocytopenia, the larger platelets have proportionately more membrane surface than the Fc receptor.

·                     Test of primary hemostasis bleeding time

o                                            This is a valuable test for disorders of primary hemostasis.

o                                            It is performed by measuring the duration required for bleeding to stop from a fresh superficial cut (1 mm deep, 1 cm long) made on the volar surface of the forearm using a template under standard conditions.

o                                            Under these conditions, the cessation of bleeding results from the formation of a primary hemostatic plug. A fairly linear correlation exists between bleeding time and platelet counts of 10,000-100,000/mL.

o                                            A prolonged bleeding time with a normal platelet count is very significant and indicates a qualitative platelet disorder.

o                                            In disorders of secondary hemostasis (eg, hemophilia A and B), bleeding time is almost invariably normal.

o                                            Bleeding time is prolonged when platelet counts indicate fewer than 75,000/mL and do not provide any further information.

o                                            This test should not be performed on patients with thrombocytopenia.

o                                            This test is highly operator-dependent and is not recommended as a routine screening test.

·                     Platelet aggregation

o                                            Platelet aggregation is measured by turbidimetric methods.

o                                            When platelets aggregate, the opalescent suspension of platelet-rich plasma becomes clearer and allows more light transmission. The extent of aggregation is determined by measuring the increase in light transmission.

o                                            Small doses of ADP (<1 mmol) induce a reversible form of platelet aggregation (primary wave), unaccompanied by thromboxane synthesis or release of intraplatelet ADP. However, with increasing doses of ADP, sufficient stimulation of platelets occurs and leads to the release of intraplatelet ADP and the synthesis of thromboxane A2 from arachidonic acid, thus resulting in more pronounced irreversible aggregation (secondary wave).

o                                            Ristocetin induces platelet aggregation by inducing von Willebrand protein binding to the platelet glycoprotein Ib complex.

o                                            Platelet aggregation tests are useful in distinguishing different disorders of platelet function. They are also particularly useful in the diagnosis of von Willebrand disease, in which ristocetin-induced platelet aggregation is defective.

Imaging Studies:

·                     Imaging studies are not necessary to diagnose uncomplicated ITPs.

·                     Rarely, platelet survival studies may be necessary to document decreased platelet survival before splenectomy in a patient with possible bone marrow hypofunction. Typically, the platelet half-life is decreased from the normal 5-7 days. A normal platelet survival curve is not consistent with increased splenic destruction.

·                     In a patient who has relapsed following splenectomy, an indium-labeled platelet imaging study is sometimes useful for localizing an accessory spleen.

Other Tests:

·                     Bone marrow examination is not necessary in most cases of platelet disorders. The isolated presence of large platelets in the peripheral blood, in the absence of any other signs of bone marrow dysfunction, is very suggestive of normal marrow activity.

·                     Bone marrow examination is necessary in patients who have an atypical course, have splenomegaly, or will undergo splenectomy.

Bone marrow examination in patients with ITP shows megakaryocytic hyperplasia (see Pic.10). Quantifying the megakaryocytes in the bone marrow is technically difficult. Usually, 2-3 megakaryocytes are present in each spicule in typical marrow. Clusters of immature megakaryocytes are often observed in patients with ITP.

evere thrombocytopenia with bleeding

o                                            A bleeding patient with a very low platelet count is a medical emergency.

o                                            The presence of hemorrhagic bullae in the buccal mucosa and retinal hemorrhages are harbingers of internal and intracranial bleeding.

o                                            Diseases that cause such severe thrombocytopenia are ITP, TTP, posttransfusion purpura, drug-induced thrombocytopenia, and aplastic anemia. Differentiating TTP from ITP is very important because platelet transfusions are contraindicated in patients with TTP and plasma exchange therapy should be initiated as soon as possible in patients with TTP.

o                                            Careful examination of the peripheral smear helps differentiate ITP from TTP. Furthermore, the presence of neurological signs, renal failure, fever, and a high LDH level also helps in the diagnosis of TTP.

o                                            The patient's medication history should be reviewed, and drug-induced thrombocytopenia should be considered if a temporal relationship exists between thrombocytopenia and drug exposure.

o                                            Patients with liver disease and those who abuse alcohol often present with severe thrombocytopenia following binge drinking. These patients may have severe thrombocytopenia resulting from splenomegaly, alcohol-induced suppression of platelet production, folate deficiency, and DIC from active liver disease.

o                                            Aplastic anemia is associated with pancytopenia, and the smear examination findings help differentiate it from ITP.

o                                            Once the diagnosis of ITP with clinically significant bleeding is established, treatment with steroids (intravenous methylprednisolone at 30 mg/kg) and IVIG should be started immediately.

o                                            Platelet transfusions are administered to patients with severe clinical bleeding, and a sustained increase in platelet counts is sometimes observed in those with ITP.

o                                            Currently, emergency splenectomy is rarely necessary and is only considered prior to an emergency operation such as evacuation of an intracranial hematoma.

Immune thrombocytopenic purpura (ITP)

Immune thrombocytopenic purpura (ITP) is a clinical syndrome in which a decreased number of circulating platelets (thrombocytopenia) manifests as a bleeding tendency, easy bruising (purpura), or extravasation of blood from capillaries into skin and mucous membranes (petechiae).

In persons with ITP, platelets are coated with autoantibodies to platelet membrane antigens, resulting in splenic sequestration and phagocytosis by mononuclear macrophages. The resulting shortened life span of platelets in the circulation, together with incomplete compensation by increased platelet production by bone marrow megakaryocytes, results in a decreased platelet count.

To establish a diagnosis of ITP, exclude other causes of thrombocytopenia, such as leukemia, myelophthisic marrow infiltration, myelodysplasia, aplastic anemia, or adverse drug reactions. Pseudothrombocytopenia due to platelet clumping is also a diagnostic consideration.

No single laboratory result or clinical finding establishes a diagnosis of ITP; it is a diagnosis of exclusion.

Pathophysiology: An abnormal autoantibody, usually immunoglobulin G (IgG) with specificity for 1 or more platelet membrane glycoproteins (GPs), binds to circulating platelet membranes.

Autoantibody-coated platelets induce Fc receptor–mediated phagocytosis by mononuclear macrophages, primarily but not exclusively in the spleen. The spleen is the key organ in the pathophysiology of ITP not only because platelet autoantibodies are formed in the white pulp but also because mononuclear macrophages in the red pulp destroy immunoglobulin-coated platelets.

If bone marrow megakaryocytes cannot increase production and maintain a normal number of circulating platelets, thrombocytopenia and purpura develop. Impaired thrombopoiesis is attributed to failure of a compensatory increase in thrombopoietin and megakaryocyte apoptosis.

Mortality/Morbidity:

·                     Hemorrhage: The primary cause of long-term morbidity and mortality is hemorrhage.

·                     Intracranial hemorrhage: The most frequent cause of death in association with ITP is spontaneous or accidental trauma-induced intracranial bleeding in patients whose platelet counts are less than 10 X 109/L (<10 X 103/mL). This situation occurs in less than 1% of patients.

·                     Treatment-related morbidity: To maintain a platelet count in a safe range in patients with chronic treatment-resistant ITP, a long-term course of corticosteroids, other immunosuppressive medications, or splenectomy may be required. In patients with this disease, morbidity and mortality can be related to treatment, reflecting the complications of therapy with corticosteroids or splenectomy.

Sex:

·                     In children, the prevalence is the same among boys and girls.

·                     In adults, women are affected approximately 3 times more frequently than men.

Age:

·                     Children may be affected at any age, but the prevalence peaks in children aged 3-5 years.

·                     Adults may be affected at any age, but most cases are diagnosed in women aged 30-40 years.

·                     Onset in a patient older than 60 years is uncommon, and a search for other causes of thrombocytopenia is warranted. The most likely causes in these persons are myelodysplastic syndromes, acute leukemia, and marrow infiltration (myelophthisis).

Clinical picture

History:

Ø    The medical history should focus on (1) factors that suggest another disease for which thrombocytopenia is a complication and (2) signs and symptoms that differentiate mild, moderate, and severe bleeding tendencies.

Ø    Other systemic illnesses

Ø    In adults, thrombocytopenic purpura may be a manifestation of systemic lupus erythematosus or acute or chronic leukemia.

Ø    Thrombocytopenic purpura may be a manifestation of a myelodysplastic syndrome, particularly in patients older than 60 years.

Ø    In young children, ITP may manifest as a primary immune deficiency syndrome.

Ø    Postviral illness

Ø    In children, most cases of ITP are acute, and onset seems to occur within a few weeks of recovery from a viral illness. The severity of symptoms of the viral illness is not correlated with the degree of thrombocytopenia.

Ø    Thrombocytopenia is a recognized complication after infection with Ebstein-Barr virus, varicella virus, cytomegalovirus, rubella virus, or hepatitis virus (A, B, or C); thought the most typical association is a vaguely defined, viral, upper respiratory infection or gastroenteritis.

Ø    Transient thrombocytopenia often follows recent immunization with attenuated live-virus vaccines.

Ø    HIV infection

Ø    Thrombocytopenia may occur during the acute retroviral syndrome coincident with fever, rash, and sore throat.

Ø    Thrombocytopenia may be a manifestation of AIDS, occurring late in the course of HIV infection.

Ø    Thrombocytopenia not uncommonly marks the onset of symptomatic HIV infection, particularly in people who abuse drugs.

Ø    Drug-induced thrombocytopenia

Ø    Regard any medication taken by a person who develops thrombocytopenia as a potential causative agent. A history of all prescription and over-the-counter medications is required to exclude drug-related thrombocytopenia.

Ø    Persons who have been sensitized (by previous exposure) to quinidine or quinine may develop immune-mediated drug purpura within hours to days of subsequent exposure. To exclude drug purpura in a person previously treated with quinidine or quinine, the history must include questions about possible exposure to over-the-counter medications, tonic water in cocktails, or bitter lemon beverages.

Ø    Investigate the records of patients who have been hospitalized and who develop acute thrombocytopenias for all of their medications that are listed and not listed in nursing charts. For example, people who are at risk for heparin-induced thrombocytopenia because of current or recent treatment with heparin may be receiving the heparin with the routine flushing of intravenous (IV) catheters, and this exposure may not be listed on the nursing medication sheet. Many catheters are also heparin impregnated, and unless checked, they can be a hidden cause of heparin-induced thrombocytopenia.

Ø    Other drugs associated with drug purpura include antibiotics (eg, cephalothins, rifampicin), gold salts, analgesics, neuroleptics, diuretics, antihypertensives, eptifibatide (Integrilin), and abciximab (ReoPro), which is a Fab fragment of the chimeric human-murine monoclonal antibody 7E3 directed against the platelet GPIIb/IIIa receptor.

Ø    Acute and chronic alcohol consumption also may be associated with thrombocytopenia. In persons with chronic liver disease, hypersplenism with secondary thrombocytopenia is not uncommon.

Physical: Similar to the medical history, focus the physical examination on (1) findings that suggest another disease for which thrombocytopenia is a complication and (2) physical signs that suggest serious internal bleeding.

Ø    General health

Ø    ITP is a primary illness occurring in an otherwise healthy person.

Ø    Signs of chronic disease, infection, wasting, or poor nutrition indicate that the patient has another illness.

Ø    Vital signs: Hypertension and bradycardia may be signs of increased intracranial pressure and evidence of an undiagnosed intracranial hemorrhage.

Ø    Skin and mucous membranes

Ø    An initial impression of the severity of ITP is formed by examining the skin and mucous membranes.

Ø    Widespread petechiae and ecchymoses, oozing from a venipuncture site, gingival bleeding, and hemorrhagic bullae indicate that the patient is at risk for a serious bleeding complication. If the patient's blood pressure was taken recently, petechiae may be observed under and distal to the area where the cuff was placed and inflated. Suction-type ECG leads may similarly induce petechiae.

Ø    Mild thrombocytopenia and a relatively low risk for a serious bleeding complication may manifest as petechiae over the ankles in patients who are ambulatory or on the back in patients who are bedridden.

Ø    Cardiovascular system: Distant low-amplitude heart sounds accompanied by jugular venous distension may be evidence of hemopericardium.

Ø    Abdomen

Ø    In an adult, hepatosplenomegaly is also atypical for ITP and may indicate chronic liver and other diseases. In fact, splenomegaly excludes the diagnosis of ITP.

Ø    Nervous system

Ø    Any asymmetrical finding of recent onset can indicate an intracranial hemorrhage.

Ø    Pupils should be equal in size and have intact extraocular muscles and symmetrical eye movements.

Ø    Balance and gait should be intact.

Ø    Funduscopic examination reveals whether the margins of the optic disc are blurred. Examine the patient for the presence of retinal hemorrhages and other evidence of increased intracranial pressure.

Causes:

 In adults, most cases of ITP are chronic, manifesting with an insidious onset, typically in middle-aged women. These clinical presentations suggest that the triggering events may be different. However, in both children and adults, the cause of thrombocytopenia (destruction of [antibody-coated] immunoglobulin-coated platelets by mononuclear macrophages) appears to be similar.

·  Autoantibody stimulation

Ø  In persons with chronic ITP, membrane GPs on the surface of platelets become immunogenic, stimulating the production of platelet autoantibodies.

Ø  In persons with acute ITP, the stimulus for autoantibody production is also unknown. Platelet membrane cryptantigens may become exposed by the stress of infection, or pseudoantigens may be formed by the passive adsorption of pathogens on platelet surfaces.

Ø  Autoantibody specificity

Ø  In persons with chronic ITP, approximately 75% of autoantibodies are directed against platelet GPIIb/IIIa or GPIb/IX GP complexes.

Ø  Presumably, the remaining 25% are directed against other membrane epitopes, including GPV, GPIa/IIa, or GPIV.

·  Role of the spleen

Ø  The spleen is the site of autoantibody production (white pulp).

Ø  It is also the site of phagocytosis of autoantibody-coated platelets (red pulp).

Ø  The slow passage of platelets through splenic sinusoids with a high local concentration of antibodies and Fc-gamma receptors on splenic macrophages lend to the uniqueness of the spleen as a site of platelet destruction.

Ø  Low-affinity macrophage receptors, Fc gamma RIIA and Fc gamma RIIIA bind immune-complexed IgG and are the key mediators of platelet clearance.

-                Platelet destruction

Ø  The mononuclear macrophage system of the spleen is responsible for removing platelets in ITP because splenectomy results in prompt restoration of normal platelet counts in most patients with ITP.

Ø  Platelets are sequestered and destroyed by mononuclear macrophages, which are neither reticular nor endothelial in origin. Therefore, the former designation of reticuloendothelial system is considered imprecise.

Ø  Immune destruction of immunoglobulin-coated platelets is mediated by macrophage IgG Fc (Fc gamma RI, Fc gamma RII, and Fc gamma RIII) and complement receptors (CR1, CR3).

Lab Studies:

·  Determination of CBC

o    The hallmark of ITP is isolated thrombocytopenia.

o    Anemia and/or neutropenia may indicate other diseases.

Ø  Peripheral blood smear

o    The morphology of RBCs and leukocytes is normal.

o    The morphology of platelets is typically normal, with varying numbers of large platelets. Some persons with acute ITP may have megathrombocytes or stress platelets, reflecting the early release of megakaryocytic fragments into the circulation.

o    If most of the platelets are large, approximating the diameter of RBCs, or if they lack granules or have an abnormal color, consider an inherited platelet disorder.

o    Clumps of platelets on a peripheral smear prepared from ethylenediaminetetraacetic acid–anticoagulated blood are evidence of pseudothrombocytopenia. The diagnosis of this type of pseudothrombocytopenia is established if the platelet count is normal when repeated on a sample from heparin-anticoagulated or citrate-anticoagulated blood.

Ø  Test for antibodies to HIV

o    In patients who have risk factors for HIV infection, a blood sample should be tested with an enzyme immunoassay for anti-HIV.

o    During the acute HIV retroviral syndrome, the results of the anti-HIV assay may be negative. In this situation, a polymerase chain reaction for HIV DNA is more reliable than the anti-HIV assay.

Ø  Test for antiplatelet antibodies

o    Assays for platelet antigen–specific antibodies, platelet-associated immunoglobulin, or other antiplatelet antibodies are available in some medical centers and certain mail-in reference laboratories.

o    The reliability of the results of a platelet antibody test is highly specific to the laboratory used. A negative antiplatelet antibody assay result does not exclude the diagnosis of ITP, and this test should not be considered part of the routine evaluation.

o    This test is not required to diagnose ITP.

Ø  Test for antinuclear antibodies

o    In selected women, the medical history may suggest a chronic, recurrent, multisystemic illness with vague, generalized signs or symptoms, such as recurrent, multiple, painful, tender, or swollen joints.

o    In such cases, a negative antinuclear antibody result is useful in diagnosing ITP if the patient's thrombocytopenia becomes chronic and resistant to treatment.

Ø  Direct antiglobulin test: If anemia and thrombocytopenia are present, a positive direct antiglobulin (Coombs) test result may help establish a diagnosis of Evans syndrome.

Ø  Helicobacter pylori testing

o    Studies from Italy and Japan indicate that many persons with ITP have H pylori gastric infections and that eradication of H pyloriresults in increased platelet counts.

o    In the United States and Spain, the prevalence of H pylori infections does not appear to be increased in persons with ITP and eradication of H pylori has not increased platelet counts.

o    Therefore, routine testing for H pylori infections in adults and children with ITP is not recommended.

Imaging Studies:

Ø  CT scanning and MRI are relatively benign and useful noninvasive imaging studies that can be used to rule out other causes of thrombocytopenia. However, they are not part of the routine evaluation of patients who may have ITP.

Ø  Promptly perform CT or MRI when the medical history or physical findings suggest serious internal bleeding.

Histologic Findings:

Bone marrow aspirate

The cellularity of the aspirate and the morphology of erythroid and myeloid precursors should be normal. The number of megakaryocytes may be increased. Because the peripheral destruction of platelets is increased, megakaryocytes may be large and immature, although in many cases the megakaryocyte morphology is normal. Older patients require a careful examination of megakaryocyte morphology to exclude an early myelodysplastic syndrome.

Bone marrow biopsy

Sections of a needle biopsy specimen or marrow clot should reveal normal marrow cellularity, without evidence of hypoplasia or increased fibrosis.

Splenic evaluation

The spleen reveals no specific findings. In adults, the microscopic finding of extramedullary hematopoiesis is atypical and indicates myeloid metaplasia. Carefully examine spleens removed from patients with ITP for a primary splenic lymphoma or granuloma or other signs of an undiagnosed infectious disease.